WO2024088865A1 - Treatment of prostate cancer by promoting pde4d7 - Google Patents

Treatment of prostate cancer by promoting pde4d7 Download PDF

Info

Publication number
WO2024088865A1
WO2024088865A1 PCT/EP2023/079089 EP2023079089W WO2024088865A1 WO 2024088865 A1 WO2024088865 A1 WO 2024088865A1 EP 2023079089 W EP2023079089 W EP 2023079089W WO 2024088865 A1 WO2024088865 A1 WO 2024088865A1
Authority
WO
WIPO (PCT)
Prior art keywords
pde4d7
resistance
peptide
expression
sequence
Prior art date
Application number
PCT/EP2023/079089
Other languages
French (fr)
Inventor
Ralf Dieter HOFFMANN
George Baillie
Original Assignee
Koninklijke Philips N.V.
The University Court Of The University Of Glasgow
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from EP22204042.0A external-priority patent/EP4360711A1/en
Application filed by Koninklijke Philips N.V., The University Court Of The University Of Glasgow filed Critical Koninklijke Philips N.V.
Publication of WO2024088865A1 publication Critical patent/WO2024088865A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to a product for use in the treatment, prevention or amelioration of prostate cancer by reducing therapy resistance in a subject in need thereof.
  • the product is used to induce the expression of PDE4D7 or promote activity of phosphodiesterase 4D7.
  • Cancer is a class of diseases in which a group of cells display uncontrolled growth, invasion and sometimes metastasis. These three malignant properties of cancers differentiate them from benign tumors, which are self-limited, do not invade or metastasize.
  • the three most commonly diagnosed cancers are prostate, lung and colorectal cancer in developed countries. Particularly prostate cancer is the most common malignancy in European males. In 2002 in Europe, an estimated 225,000 men were newly diagnosed with prostate cancer and about 83,000 died from this disease.
  • Prostate cancer for example, is traditionally diagnosed via the serum level of prostatespecific antigen (PSA).
  • PSA prostatespecific antigen
  • PSA is not prostate cancer-specific and can be raised in other circumstances, leading to a large number of false-positive s (cancer is not found in around 70% of men with raised PSA levels who undergo biopsy).
  • cancer is not found in around 70% of men with raised PSA levels who undergo biopsy.
  • Androgen ablation therapy causes remission in 80-90% of patients undergoing therapy, resulting in a median progression-free survival of 12 to 33 months. After remission, an androgenindependent phenotype typically emerges, wherein the median overall survival is 23-37 months from the time of initiation of androgen ablation therapy. How androgen-independence is established and how it reestablishes progression is unclear. Androgen ablation therapy may for example include taking antiandrogen drugs.
  • US 2012/129788 Al relates to the 4D7 isoform of PDE and teaches that tumors have increased PDE4D7 expression, and this can be used for diagnosis purposes.
  • EP3434787 Al teaches that expression of PDE4D7 is reduced in hormone resistance and diagnosis using PDE4D7 expression levels.
  • EP3303618 Al relates to methods for determining gene expression signatures for diagnosing patients with prostate cancer, e.g., to establish a prognosis for such patients, for determining the predisposition of said patient to develop aggressive or indolent disease, for example after primary treatment, and/or for identification and stratification of patients for available therapies.
  • Boettcher et al. (ONCOTARGET, vol. 7, no. 43, 25 October 2016 (2016-10-25), pages 70669-70684) describes that PDE4D isoform composition is deregulated in primary prostate cancer and indicative for disease progression and development of distant metastases.
  • the invention relates to a product for use in the treatment, prevention or amelioration of prostate cancer by reducing therapy resistance in a subject in need thereof, wherein the product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7 and is selected from: a polynucleotide encoding a peptide having a sequence according to SEQ ID NO: 2 or a variant thereof, wherein the variant is a polynucleotide encoding a peptide having a sequence with at least 90% sequence homology with SEQ ID NO: 2 and wherein the peptide has phosphodiesterase activity; or a polynucleotide comprising a nucleotide sequence as defined in SEQ ID NO: 1 or a variant thereof, wherein the variant is a polynucleotide comprising a nucleotide sequence with at least 90% sequence homology to SEQ ID NO: 1 and wherein the polynucleotide encodes a peptide that has
  • Ri is H, (Ci-4)alkyl or (Ci-4)alkyloxy;
  • R2 and Re are independently selected from H and;
  • Rs, R4 and R5 are independently selected from H, halogen, CN, (Ci-4)alkyl and (Ci-4)alkyloxy, the (Ci- 4)alkyl and (Ci-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
  • R7, Rs, and Rio are independently selected from H and F;
  • Rs is selected from (Ci-4)alkyl, (Ci-4)alkyloxy, CN and halogen, the (Ci-4)alkyl and (Ci-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros; or a peptide having a sequence comprising or consisting a sequence selected from SEQ ID Nos: 24, 25, 26 or 27, or a polynucleotide encoding a peptide having a sequence comprising or consisting a sequence selected from SEQ ID Nos: 24, 25, 26 or 27.
  • the invention in a second aspect relates to a compound for use in the treatment, prevention or amelioration of prostate cancer in a subject in need thereof, wherein the compound is an ATR inhibitor or an metabolic inhibitor, the use comprising administering the compound if: high expression of PDE4D7 is observed in a tumor; low expression of PDE4D7 is observed but PDE4D7 activity cannot be sufficiently increased to re-sensitize tumors to treatment with inhibitors; or in metastatic tumors.
  • Fig. 1 Generation of PDE4D7-knockdown LNCaP cell lines and differences in PCa- related genes.
  • D Heatmap of z-scores of top differentially expressed genes between WT and Pl LNCaPs via RNAseq.
  • E Log fold change (FC) of top altered genes in PCa-related pathways.
  • Fig. 2 PDE4D7 knockdown in LNCaP cells enhances growth and confers resistance to enzalutamide.
  • A) Real-time proliferation of LNCaP WT, shRNA-scrambled (SC2) and shRNA-PDE4D7 knock-down (Pl) cells. Slope analysis via linear regression.
  • B & C Real-time analysis of LNCaP WT (B) and shRNA-PDE4D7 knock-down (Pl) cells (C) with enzalutamide (OpM - 30pM) or 0.1% DMSO.
  • E) Cyclin Bl and F) Cyclin DI protein expression western blot and quantification in WT and Pl LNCaPs. Error bars represent mean +/- SEM, N 3, ** p ⁇ 0.01, **** pO.OOOl).
  • Fig. 3 PDE4D7-knockdown in LNCaP cells enhances resistance to olaparib.
  • a & B Real-time proliferation of LNCaP WT (A) and shRNA-PDE4D7 knock-down (Pl) cells following treatment with olaparib (OpM - 30pM) or 0.1% DMSO. Normalised cell index to timepoint of treatment. Slope analysis via linear regression between 0-30h.
  • Fig. 4 DNA damage induction and repair agents affect both WT and PDE4D7- knockdown LNCaPs (Pl).
  • DMSO control (0.1%)
  • DOC 5 5 pM docetaxel
  • DOC 7.5 7.5 pM docetaxel
  • DOC 10 10 pM docetaxel
  • DOC 25 25 pM docetaxel
  • treatment on WT A) or Pl (B) LNCaPs, or Pl and
  • C P1-PDE4D7+/+ (after transient PDE4D7 reexpression) LNCaPs including the slope analysis 0-48h since treatment.
  • Fig. 5 PDE4D7-knockdown alters LNCaP response to various compounds.
  • A) Glucocorticoid receptor (GR) protein expression western blot and quantification between WT and PDE4D7-knock-down Pl LNCaPs.
  • GR Glucocorticoid receptor
  • D & E Real-time proliferation of WT (D) and PDE4D7-knock-down Pl (E) LNCaPs upon treatment with l-10pM mebendazole or 0.1% DMSO including slope analysis 0-48h post-treatment.
  • Fig 6 A & B) Real-time growth analysis of WT (A) and PDE4D7-knock-down Pl (B) cells upon treatment with metformin (0-25 pM) including slope analysis from 0-48h post-treatment.
  • C & D Real-time growth analysis of WT (C) and PDE4D7-knock-down Pl (D) cells upon treatment with PDE4 activator ML-R2 (l-10pM) including slope analysis from 0-48 post-treatment.
  • Fig. 7 Difference in PDE4D isoform mRNA expression between knockdown LNCaP cell lines.
  • RT-qPCR of various PDE4D knockdown LNCaP cell lines shows fold changes in gene expression relative to WT (2-AACt) for PDE4D5 (A), PDE4D7 (B) and PDE4D9 (C.)
  • Fig. 9 Re-expression of PDE4D7 reduces growth.
  • A Real-time growth analysis of PDE4D7-knowdown Pl and PDE4D7-knowdown with stable re-introduction of PDE4D7 P1-4D7+ cells.
  • B Slope analysis from 0-72 post-onset.
  • Fig. 10 Testing of the PDE4 activating compound MR-L2 on the real-time proliferation of prostate cancer cells; (i) real-time proliferation; (ii) slope analysis of real-time proliferation.
  • Fig. 11 Testing of the PARP inhibitor olaparib on the real-time proliferation of prostate cancer cells.
  • P1+4D7 DMSO LNCaP PDE4D7 knockdown cell line Pl with transient re-expression of PDE4D7 DMSO control (0.1%);
  • Fig. 12 Ratio of PARP and cleaved PARP (PARPc) by western blot analysis in LNCaP WT and LNCaP PDE4D7 knockdown cell line Pl upon incubation with 0.1% DMSO (control), 10 pM Olaparib, and 10 nM docetaxel including quantitation of western blot bands normalised to DMSO.
  • Fig. 13 Survival analysis of the PDE4D7 Score to prostate cancer specific mortality (PCSM) after start of salvage radiation therapy (SRT).
  • PCSM prostate cancer specific mortality
  • SRT salvage radiation therapy
  • Two cut-offs for the pPDE4D7 score were defined by AUROC (Area under the ROC curve) analysis with 5-year prostate cancer specific death after start of SRT as the dependent variable and the pPDE4D7 score as the independent variable.
  • One cut-off (pPDE4D7>0.2) was defined as the point in the AUROC with maximum sensitivity and specificity.
  • the cut-off was defined as max sensitivity (pPDE4D7>0.87).
  • (A) Kaplan Meier survival analysis of the time to prostate cancer specific death after start of SRT in N 348 clinical high risk prostate cancer patient cohort. Patient groups are stratified according to their PDE4D7 Score with high scores being associated with less risk of death due to prostate cancer compared to low PDE4D7 scores.
  • (B) Kaplan Meier survival analysis of the time to death due to all causes after start of SRT in N 348 clinical high risk prostate cancer patient cohort.
  • (C) Kaplan Meier survival analysis of the time to prostate cancer specific death after start of SRT in N 348 clinical high risk prostate cancer patient cohort. Patient groups are stratified according to the EAU-BCR Risk Score which stratifies patients into a low vs. a high-risk class.
  • (D) Kaplan Meier survival analysis post-SRT of the time to prostate cancer specific death after start of ADT in a N 179 prostate cancer patient cohort.
  • (E) Kaplan Meier survival analysis post-SRT of the time to death due to all causes after start of ADT in a N 179 prostate cancer patient cohort. Logrank p-values are indicated.
  • Fig. 15 PDE4D7 knockdown enhances growth and confers enzalutamide resistance, which is rescued upon PDE4D7 re-expression.
  • A Real-time growth analysis of P1-TET4D7 +- doxycycline induction of PDE4D7 alone or
  • B in response to enzalutamide lOpM.
  • Fig. 16 Expression boxplot of the BRCA2 gene in PDE4D7 expression classes in human clinical patient samples. The expression of each gene is provided after TPM calculation based on the RNAseq count data.
  • the PDE4D7 classes represent different categories of PDE4D7 expression based on the PDE4D7 score (see references) where PDE4D7_classl (most left-hand box) represents the lowest PDE4D7 scores (i.e., lowest PDE4D7 expression) while PDE4D7_class4 (most right-hand box) represents the highest PDE4D7 scores (i.e., highest PDE4D7 expression).
  • the median of the expression per group is indicated by the bar within each box.
  • the red cross represents the mean expression value per group.
  • the circles represent outlier expression values.
  • the p- values were calculated by use of ANOVA Kruskal-Wallis test and represent a significant change in expression over the four PDE4D7 classes.
  • Fig. 18 Influence of PDE4D7 expression on cAMP dynamics within LNCaPs.
  • FRET ratio (YFP/CFP) at cytosol of WT (A) or Pl (C) LNCaPs, or at membrane of WT (B) or Pl (D) LNCaPs upon treatment with rolipram or combined treatment of forskolin + IBMX.
  • the terms “about” and “approximately” denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question.
  • the term typically indicates a deviation from the indicated numerical value of ⁇ 20%, preferably ⁇ 15%, more preferably ⁇ 10%, and even more preferably ⁇ 5%.
  • “About” and “approximately” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • Antagonist and “inhibitor” are used interchangeably, and they refer to a compound or agent having the ability to reduce or inhibit a biological function of a target protein or polypeptide, such as by reducing or inhibiting the activity or expression of the target protein or polypeptide. Accordingly, the terms “antagonist” and “inhibitor” are defined in the context of the biological role of the target protein or polypeptide. An inhibitor need not completely abrogate the biological function of a target protein or polypeptide, and in some embodiments reduces the activity by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • While some antagonists herein specifically interact with (e.g., bind to) the target, compounds that inhibit a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target protein or polypeptide are also specifically included within this definition.
  • Non-limiting examples of biological activity inhibited by an antagonist include those associated with the development, growth, or spread of a tumor, or an undesired immune response as manifested in autoimmune disease.
  • Anti-cancer effect This refers to the effect a therapeutic agent has on cancer, e.g., a decrease in growth, viability, or both of a cancer cell.
  • the IC50 of cancer cells can be used as a measure the anti-cancer effect.
  • IC50 refers to a measure of the effectiveness of a therapeutic agent in inhibiting cancer cells by 50%.
  • “Alleviating cancer” The term, in the context of specific cancers and/or their pathologies, refers to degrading a tumor, for example, breaking down the structural integrity or connective tissue of a tumor, such that the tumor size is reduced when compared to the tumor size before treatment. “Alleviating” metastasis of cancer includes reducing the rate at which the cancer spreads to other organs.
  • compositions “Compositions”, “products” or “combinations”: These encompass those compositions suitable for various routes of administration, including, but not limited to, intravenous, subcutaneous, intradermal, subdermal, intranodal, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral or mucosal application.
  • the compositions, formulations, and products according to the disclosure invention normally comprise the drugs/compound/inhibitor (alone or in combination) and one or more suitable pharmaceutically acceptable excipients or carriers.
  • Compound 1 refers to the use of more than one compound or agent to treat a particular disorder or condition.
  • Compound 1 may be administered in combination with at least one additional therapeutic agent.
  • in combination with it is not intended to imply that the other therapy and Compound 1 must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of this disclosure.
  • Compound 1 can be administered concurrently with, prior to (e.g.
  • each therapeutic agent will be administered at a dose and/or on a time schedule determined for that particular agent.
  • the other therapeutic agent can be administered with Compound 1 herein in a single composition or separately in a different composition. Higher combinations, e.g., triple therapy, are also contemplated herein.
  • the term “comprising” is not limiting.
  • the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.
  • the terms “first”, “second”, “third” or “(a)”, “(b)”, “(c)”, “(d)” etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
  • first”, “second”, “third” or “(a)”, “(b)”, “(c)”, “(d)” etc. relate to steps of a method or use there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.
  • the term "at least” a particular value means that particular value or more.
  • “at least 2" is understood to be the same as “2 or more” i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ... , etc.
  • the term “at most” a particular value means that particular value or less.
  • “at most 5" is understood to be the same as "5 or less” i.e., 5, 4, 3, ... .-10, -11, etc.
  • the word “comprise” or variations thereof such as “comprises” or “comprising” will be understood to include a stated element, integer or step, or group of elements, integers or steps, but not to exclude any other element, integer or steps, or groups of elements, integers or steps.
  • the verb “comprising” includes the verbs “essentially consisting of’ and “consisting of’.
  • conventional techniques refers to a situation wherein the methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker.
  • the practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.
  • identity refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (Computational Molecular Biology, Lesk, A. M., ED., Oxford University Press, New York, 1988; Biocomputing: Informatics And Genome Projects, Smith, D. W., ED., Academic Press, New York, 1993; Computer Analysis Of Sequence Data, Part I, Griffin, A. M., And Griffin, H.
  • nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a certain sequence
  • the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference amino acid sequence.
  • nucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted and/or substituted with another nucleotide, and/or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID NO: X is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: X.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • in vitro refers to experimentation or measurements conducted using components of an organism that have been isolated from their natural conditions.
  • nucleic acid As used herein, the term “ex vivo” refers to experimentation or measurements done in or on tissue from an organism in an external environment with minimal alteration of natural condition.
  • nucleic acid As used herein, the term “nucleic acid”, “nucleic acid molecule” and “polynucleotide” is intended to include DNA molecules and RNA molecules.
  • a nucleic acid (molecule) may be singlestranded or double-stranded, but preferably is double-stranded DNA.
  • sequence when referring to nucleotides, or “nucleic acid sequence”, “nucleotide sequence” or “polynucleotide sequence” refer to the order of nucleotides of, or within, a nucleic acid and/or polynucleotide.
  • a first nucleic acid sequence may be comprised within or overlap with a further nucleic acid sequence.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo-, sports-, or pet-animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, bears, and so on.
  • a subject may be alive or dead. Samples can be taken from a subject post-mortem, i.e. after death, and/or samples can be taken from a living subject.
  • treatment refers to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit.
  • therapeutic benefit is meant eradication or amelioration or reduction (or delay) of progress of the underlying disease being treated.
  • a therapeutic benefit is achieved with the eradication or amelioration or reduction (or delay) of progress of one or more of the physiological symptoms associated with the underlying disease such that an improvement or slowing down or reduction of decline is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disease.
  • a portion of this invention contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction).
  • copyright protection such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.
  • the copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent invention, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.
  • Phosphodiesterases are a superfamily of enzymes which play the fundamental role of hydrolysing cAMP, leading to its degradation and termination of cell signal transduction. These enzymes define and regulate intracellular cAMP gradients around specific signalling complexes, with each specific isoform having unique functional roles.
  • PDE4 family of enzymes, which is made up of over 25 different isoforms, many of which have important, non-redundant functions. Often, the function of a particular PDE4 isoform is conferred by its unique N-terminal, which acts as a “postcode” to anchor PDE4 enzymes to discrete intracellular domains where they sculpt signal-specific cAMP gradients. PDE4s also contain a catalytic unit and regulatory domains termed “upstream conserved regions one and two” (UCR1/2) which are highly conserved throughout the isoforms. All long-form PDE4s, such as for example PDE4D7, contain UCR1, which contains a PKA motif that becomes phosphorylated during conditions of raised cAMP. Such an action serves to activate PDE4 and rapidly reduce the local concentration of cAMP. This feedback loop underpins the transient nature of cAMP signals and ensures a rapid but fleeting response to activation of Gas-coupled receptors.
  • Deregulated cAMP signalling is associated with various diseases including cancer, with the PDE4D sub-family of particular interest in prostate cancer (PCa).
  • PCa prostate cancer
  • PDE4D7 a long PDE4D isoform variant, localises to the sub-plasma membrane in PCa cells and its expression inversely correlates with disease progression.
  • PDE4D7 mRNA is significantly downregulated from an androgen sensitive (AS) to androgen insensitive (Al) disease phenotype, implicating PDE4D7 in PCa development and progression.
  • AS androgen sensitive
  • Al androgen insensitive
  • PDE4D7 expression and presence of the TMPRSS2-ERG gene fusion the most common gene rearrangement in PCa which leads to overexpression of ERG and subsequent enhanced PCa aggressiveness.
  • the inventors describe data obtained from prostate cancer cells in which expression of PDE4D7 has been specifically reduced using shRNA mediated knockdown. Surprisingly the inventors found that re-expressing PDE4D7 in these knockdown cells reduced proliferation. Furthermore expression of PDE4D7 was able to reduce resistance to drug treatments such as antiandrogen resistance, PARP inhibitor resistance or chemotherapy resistance. Based on these data the inventors concluded that increasing expression or increasing PDE4D7 phosphodiesterase activity reduces therapy resistance and may be used to treat therapy resistant prostate cancers.
  • the invention relates to a product for use in the treatment, prevention or amelioration of prostate cancer by reducing therapy resistance in a subject in need thereof, wherein the product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7.
  • the invention relates to a method of treating, preventing or ameliorating prostate cancer in a subject in need thereof, the method comprising administering to the subject product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7 and is selected from: a polynucleotide encoding a peptide having a sequence according to SEQ ID NO: 2 or a variant thereof, wherein the variant is a polynucleotide encoding a peptide having a sequence with at least 90% sequence homology with SEQ ID NO: 2 and wherein the peptide has phosphodiesterase activity; or a polynucleotide comprising a nucleotide sequence as defined in SEQ ID NO: 1 or a variant thereof, wherein the variant is a polynucleotide comprising a nucleotide sequence with at least 90% sequence homology to SEQ ID NO: 1 and wherein the polynucleotide encodes a peptide that has phosphodiesterase activity
  • Ri is H, (Cl-4)alkyl or (Cl-4)alkyloxy;
  • R2 and Re are independently selected from H and;
  • Rs, R4 and R5 are independently selected from H, halogen, CN, (Ci-4)alkyl and (Ci-4)alkyloxy, the (Ci- 4)alkyl and (Ci-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
  • R7, Rs, and Rio are independently selected from H and F;
  • Rs is selected from (Cl-4)alkyl, (Cl-4)alkyloxy, CN and halogen, the (Cl-4)alkyl and (Cl-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros; or a peptide having a sequence comprising or consisting a sequence selected from SEQ ID Nos: 24, 25, 26 or 27, or a polynucleotide encoding a peptide having a sequence comprising or consisting a sequence selected from SEQ ID Nos: 24, 25, 26 or 27.
  • phosphodiesterase 4D7 for use as a therapeutic strategy.
  • PDE4D7 phosphodiesterase 4D7
  • the term “phosphodiesterase 4D7” or “PDE4D7” relates to the splice variant 7 of the human phosphodiesterase PDE4D, i.e. the human phosphodiesterase PDE4D7 gene, preferably to the sequence as defined in Genbank Accession No: AF536976 (version AF536976.1, GE22901883 as of 3 Mar.
  • nucleotide sequence as set forth in SEQ ID NO: 1 which corresponds to the sequence of the above indicated Genbank Accession number of the PDE4D7 transcript and corresponding protein accession number AAN10118 (version AAN10118.1)
  • SEQ ID NO: 2 which corresponds to the sequence of the above indicated Genbank Accession number of the PDE4D7 polypeptide encoded by the PDE4D7 transcript.
  • the term also comprises nucleotide sequences showing a high degree of homology to PDE4D7, e.g.
  • nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 1, or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 2, or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 2, or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO
  • human phosphodiesterase PDE4D7 gene relates to the gene encoding phosphodiesterase 4D.
  • the term relates to a gene expressing phosphodiesterase 4D as splice variant 7, e.g. the specific exon combination as defined in Genbank Accession No: AF536976 (version AF536976.1, GE22901883 as of 3 Mar. 2009) or as set forth in SEQ ID NO: 1.
  • the term also relates to DNA molecules derived from mRNA transcripts encoding phosphodiesterase 4D spliced as variant 7, preferably cDNA molecules.
  • phosphodiesterase 4D7 relates to the 4D7 splice variant encoded protein as defined herein above.
  • the term relates to a protein encoded by the phosphodiesterase 4D gene as splice variant 7, e.g. the specific exon combination as defined in Genbank Accession No: AF536976 (version AF536976.1, GE22901883 as of 3 Mar. 2009) or as set forth in SEQ ID NO: 2.
  • the term also relates to proteins or polypeptides derived from the protein encoded by the PDE4D7 gene and which have been modified by addition of additional amino acid(s) or other modifications such as sugar residues, tags, phosphorylation, acetylation, methylation, ubiquitination or other modifications, or has been modified by the removal of one or more amino acids such as but not limited to a signal peptide.
  • the present disclosure broadly defines a product for inducing the expression of PDE4D7 or promoting the activity of phosphodiesterase 4D7.
  • the product is a compound or composition.
  • the term compound may refer to a chemical compound (also referred to as “small molecule therapy”), a biological such but not limited to a polynucleotide, a polypeptide, or a sugar polymer.
  • composition may refer to a mixture of compounds or a combination of a compound with adjuvants.
  • a composition may be a solution or a dry composition.
  • the product is selected from: a gene therapy for inducing PDE4D7 expression; a compound that induces PDE4D7 expression; a compound directly stimulating or promoting phosphodiesterase 4D7 activity; a compound indirectly stimulating or promoting phosphodiesterase 4D7 activity; an inhibitor of a transcriptional inhibitor of PDE4D7; an inhibitor of an inhibitor of phosphodiesterase 4D7 enzymatic activity mRNA encoding a phosphodiesterase 4D7 protein; a phosphodiesterase 4D7 protein; a phosphodiesterase 4D7 activity promoting amyloid beta peptide.
  • the invention describes a polynucleotide encoding a peptide having a sequence according to SEQ ID NO: 2 or a variant thereof, wherein the variant is a polynucleotide encoding a peptide having a sequence with at least 90% sequence homology with SEQ ID NO: 2 and wherein the peptide has phosphodiesterase activity.
  • SEQ ID NO: 2 describes the amino acid sequence of the PDE4D isoform 7. Therefore a peptide comprising or consisting of a amino acid sequence as defined in SE QID NO: 2 (or a variant thereof as defined herein) can directly be introduced in a cell.
  • Methods for introducing a peptide in a cell are known to the skilled person and include but are not limited to using cell penetrating peptides and nanoparticles.
  • the invention describes a polynucleotide comprising a nucleotide sequence as defined in SEQ ID NO: 1 or a variant thereof, wherein the variant is a polynucleotide comprising a nucleotide sequence with at least 90% sequence homology to SEQ ID NO: 1 and wherein the polynucleotide encodes a peptide that has phosphodiesterase activity.
  • a compound as defined in formula I is a compound as defined in formula I
  • Ri is H, (Cl-4)alkyl or (Cl-4)alkyloxy;
  • R2 and Re are independently selected from H and;
  • Rs, R4 and R5 are independently selected from H, halogen, CN, (Ci-4)alkyl and (Ci-4)alkyloxy, the (Ci- 4)alkyl and (Ci-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
  • R7, Rs, and Rio are independently selected from H and F;
  • Rs is selected from (Cl-4)alkyl, (Cl-4)alkyloxy, CN and halogen, the (Cl-4)alkyl and (Cl-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros.
  • a gene therapy for inducing PDE4D7 expression refers to a method to introduce a nucleotide encoding the phosphodiesterase 4D7 isoform in a cell, preferably a cell of a subject having prostate cancer, more preferably in a prostate cancer cell or a metastasis thereof.
  • the nucleotide may for example be a viral vector or a non-viral vector, however it is understood that any nucleotide which can be introduced in a cell of a patient and express PDE4D7 may be used.
  • the nucleotide may also be an mRNA encoding PDE4D7 or a variant of PDE4D7.
  • Nucleotides such as mRNA may be expressed in a tumor cell in vivo using lipid nanoparticles (LNP). Suitable LNP formulations are known to the skilled person.
  • Viral vectors are a known strategy for gene therapy, typically viral-vector gene therapies use modified viruses as drug-delivery vehicles to introduce specific DNA or RNA sequences into cells.
  • the vector is packaged using the viral proteins allowing infection of cells and expression of its viral genome.
  • the viral vector is modified such that no new viral particles can be produced upon infection of the target cell.
  • Non limiting examples include retroviruses (RV), adenoviruses (AV), adeno- associated viruses (AAV), lentiviruses (LV), and herpes simplex viruses (HSV).
  • RSV retroviruses
  • AV adenoviruses
  • AAV adeno- associated viruses
  • LV lentiviruses
  • HSV herpes simplex viruses
  • Other ways to use viral particles or viral vectors to introduce the PDE4D7 encoding nucleotide are known to the person skilled in the art and also envisaged to be encompassed by the invention.
  • a non-viral vector may be used.
  • These vectors typically contain a promotor to drive expression of the relevant construct (e.g. PDE4D7), and typically require some means to introduce the vector into the target cell.
  • Means to deliver a non-viral vector to a target cell in a subject are known to the skilled person, and for example reviewed in Ramamoorth M, Narvekar A. Non viral vectors in gene therapy- an overview. J Clin Diagn Res. 2015 Jan;9(l):GE01-6 (hereby incorporated by reference in its entirety).
  • nanoparticles can be used to encapsulate the vector.
  • Non limiting examples are lipid based nanoparticles, peptide based nanoparticles, cationic lipid based nanoparticles, apolipoprotein based nanoparticles, (synthetic) polymer based nanoparticles such as polyethylenimine (PEI), chitosan, polylactate, polylactide, polyglucoside, denriomer or polymethracylate based nanoparticles.
  • PEI polyethylenimine
  • chitosan polylactate
  • polylactide polyglucoside
  • denriomer or polymethracylate based nanoparticles When used herein a nanoparticle is a small particle which can be used a sa carrier to deliver a cargo (payload) in a patient.
  • the cargo is the product as broadly defined herein.
  • a nanoparticle can be used to deliver the product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7 to a site in patient, e.g. a cell, tissue or organ.
  • a site in patient e.g. a cell, tissue or organ.
  • Other ways to use non- viral vectors to introduce the PDE4D7 encoding nucleotide are known to the person skilled in the art and also envisaged to be encompassed by the invention.
  • the gene therapy is selected from: a viral vector capable of expressing PDE4D7 in a subject or a non-viral vector capable of expressing PDE4D7 in a subject.
  • the product is delivered using a nanoparticle.
  • vector is used to indicate any particle (e.g., a plasmid, a cosmid, a Lambda phage) used as a vehicle to artificially carry a foreign nucleic sequence - usually DNA - into another cell, where it can be replicated and/or expressed.
  • particle e.g., a plasmid, a cosmid, a Lambda phage
  • a compound that induces PDE4D7 expression is intended to refer to any biological or chemical compound that is able to increase the expression of PDE4D7. This may for example be achieved by promoting transcription of the PDE4D7 gene or inhibiting degradation of the phosphodiesterase 4D7 isoform protein.
  • the compound may engage a signal transduction pathway to indirectly promote transcription of the PDE4D7 or directly interact with the promoter region of the genomic DNA to promote transcription of the PDE4D7 isoform.
  • promoting transcription of PDE4D7 refers to increasing the transcription of PDE4D7 resulting in a higher amount of PDE4D7 mRNA transcripts, and/or preferably in a higher amount of phosphodiesterase 4D7 isoform protein in the cell.
  • the promoting may be specific for PDE4D7, specific for all PDE4D isoforms, specific for all PDE4 or even all PDE family members. In other words, increasing expression of PDE4D7 does not exclude that other PDE4D isoforms, other PDE4 family members or even other PDE family members are also increased in expression.
  • a compound directly stimulating or promoting phosphodiesterase 4D7 activity is intended to refer to a compound which directly engages with the phosphodiesterase 4D7 isoform protein enzymatic activity.
  • PDE proteins such as the PDE4D7 protein can exist in an active and inactive conformation, wherein activity can be induced by other compounds through interaction with the PDE protein, for example by promoting an active conformation of the protein or by blocking or preventing binding of inhibitors, or by modifying the protein to promote activity (for example by phosphorylation or other known protein modifications).
  • enzymatic activity when referring to a phosphodiesterase refers to catalysis of the hydrolysis of cAMP and/or cGMP.
  • a compound indirectly stimulating or promoting phosphodiesterase 4D7 activity is intended to refer to a compound which indirectly engages with the phosphodiesterase 4D7 isoform protein enzymatic activity.
  • compounds may induce activators of PDE4D7 or block inhibitors form PDE4D7 activity and thus indirectly affect the protein’s enzymatic activity.
  • the person skilled in the art is aware of methods to determine PDE or specifically PDE4D7 activity. For example, commercial products are available to assay for PDE activity, and methods have for example been described in Blair et al. Measuring cAMP Specific Phosphodiesterase Activity: A Two-step Radioassay. Bio Protoc. 2020 Apr 5;10(7):e3581, hereby incorporated in its entirety by reference.
  • an inhibitor of a transcriptional inhibitor of PDE4D7 refers to a compound capable of blocking an inhibitor of PDE4D7 gene transcription.
  • the inbitior of PDE4D7 gene transcription may be a direct inhibitor capable of binding the genomic DNA and preventing or reducing gene transcription or an indirect inhibitor which through downstream actions reduces or inhibits transcription of the PDE4D7 gene.
  • an inhibitor of an inhibitor of phosphodiesterase 4D7 enzymatic activity refers to a compound that can block an inhibitor of phosphodiesterase isoform 4D7 protein activity.
  • the compound may function by degrading the inhibitor, preventing the inhibitor from engaging with PDE4D7 or by inhibiting the activity of the inhibitor.
  • mRNA encoding a phosphodiesterase 4D7 protein refers to a RNA molecule from which the protein corresponding to SEQ ID NO: 2 or a substantially similar protein can be translated.
  • the mRNA molecule generally comprises non translated elements such as 5’ and 3’ UTRs. It is anticipated that by direct introduction of PDE4D7 mRNA into the target cell (e.g. a prostate cancer cell in a subject) PDE4D7 can by upregulated. Method of delivering mRNA to a target cell are known to the skilled person, for example using nanobodies as described above.
  • a phosphodiesterase 4D7 protein refers to a compound comprising PDE4D7 protein or a biosimilar, or a constitutively active variant thereof, having phosphodiesterase activity. It is anticipated that by direct introduction of PDE4D7 protein into the target cell (e.g. a prostate cancer cell in a subject) PDE4D7 activity can be increased. Method of delivering protein to a target cell are known to the skilled person, for example using nanobodies as described above.
  • a phosphodiesterase 4D7 activity promoting peptide refers to a peptide that is able to increase activity of the PDE4D7 enzyme.
  • Non limiting examples are Amyloid beta (Abeta) peptides, which have been demonstrated to specifically increase long PDE4D isoforms such as PDE4D7.
  • Amyloid beta also known as Ap or Abeta, refers to peptides of 37-49 amino acids (Chen et al. Acta Pharmacol Sin 38, 1205-1235 (2017)) that are the main component of the amyloid plaques found in the brains of people with Alzheimer's disease.
  • Amyloid beta peptide (A ) is produced through the proteolytic processing of a transmembrane protein, amyloid precursor protein (APP), by P- and y-secretases.
  • APP amyloid precursor protein
  • the phosphodiesterase 4D7 activity promoting peptide is preferably a Abeta peptide or derivative thereof.
  • a Abeta peptide or derivative thereof is characterized as a peptide having a length of 16 to 50 amino acids and comprising an amino acid sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence defined by SEQ ID NO: 24 below (Abeta core peptide 1), more preferably comprising an amino acid sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence defined by SEQ ID NO: 25 below (Abeta core peptide 2).
  • Abeta 42 SEQ ID NO: 26
  • Abeta 40 SEQ ID NO: 27
  • the Abeta peptide or derivative thereof is characterized as a peptide having a length of 42 to 50 amino acids and comprising an amino acid sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence defined by SEQ ID NO: 26, or a peptide having a length of 40 to 50 amino acids and comprising an amino acid sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence defined by SEQ ID NO: 27.
  • the inventors provide experimental data strongly suggesting that increasing PDE4D7 expression or activating phosphodiesterase 4D7 in a cell may help to overcome therapy resistance, or at least makes therapy resistant tumor cells more responsive to therapeutics, such as, among others, antiandrogens, PARP inhibitors and taxanes. Therefore in an embodiment the treatment, prevention or amelioration comprises reducing a therapy resistance.
  • reducing therapy resistance refers reducing or avoiding resistance to said therapy which has developed or may develop in a cancer cell.
  • co-adminstering the compound (product) that induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7 together with a therapy will unexpectedly increase the effectiveness of the therapy, in addition to the beneficial effects observed from the compounds derscibed herein.
  • the therapy resistance is antiandrogen resistance.
  • Antiandrogens may be androgen receptor antagonists.
  • steroidal antiandrogens comprise 17a-Hydroxy progesterone derivatives such as, Chlormadinone acetate, Cyproterone acetate, Megestrol acetate, Osaterone acetate; 19-Norprogesterone derivatives such as Nomegestrol acetate; 19- Nortestosterone derivatives such as Dienogest, Oxendolone; 17a-Spirolactone derivatives such as Drospirenone, Spironolactone, Others such as Medrogestone.
  • Non-limiting examples of nonsteroidal antiandrogens comprise Bicalutamide, Flutamide, Nilutamide, Apalutamide, Darolutamide, Enzalutamide, Proxalutamide, Cimetidine and Topilutamide.
  • Antiandrogens may be androgen synthesis inhibitors.
  • Non-limiting examples of CYP17A1 inhibitors such as Abiraterone acetate, Ketoconazole, Seviteronel; CYP11A1 (P450scc) inhibitors such as Aminoglutethimide; 5a-Reductase inhibitors such as Alfatradiol, Dutasteride, Epristeride, Finasteride, Saw palmetto extract.
  • Antiandrogens may be antigonadotropins.
  • Non-limiting exmaples are estrogens such as estradiol (and its esters), ethinylestradiol, conjugated estrogens, diethylstilbestrol; GnRH analogues such as GnRH agonists like goserelin, leuprorelin, GnRH antagonists like cetrorelix; Progestogens such as chlormadinone acetate, cyproterone acetate, gestonorone caproate, medroxyprogesterone acetate, megestrol acetate.
  • the therapy resistance is resistance to an androgen receptor antagonist, an androgen synthesis inhibitor or an antigonadotropin, preferably an androgen receptor antagonist, an androgen synthesis inhibitor or an antigonadotropin as listed above.
  • the therapy resistance is resistance to Enzalutamide, Flutamide, Nilutamide, Bicalutamide, Topilutamide, Apalutamide, Darolutamide, Cimetidine, ketoconazole, Proxalutamide (GT-0918), Seviteronel (VT-464), or Abiraterone.
  • the therapy resistance is resistance to Enzalutamide.
  • the therapy resistance is PARP inhibitor resistance.
  • the PARP inhibitor resistance may be resistance to Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, Pamiparib (BGB-290), Rucaparib, CEP 9722, E7016, Iniparib or 3 -Aminobenzamide. Therefore in an embodiment the therapy resistance is resistance to a PARP inhibitor as listed above. In an embodiment the therapy resistance is resistance to a PARP inhibitor selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, Pamiparib (BGB-290), Rucaparib, CEP 9722, or E7016. In an embodiment the therapy resistance is resistance to Olaparib.
  • the therapy resistance is resistance to a chemotherapeutic agent.
  • a chemotherapeutic agent may be an alkylating agent.
  • alkylating agents are Cyclophosphamide, Mechlorethamine, Chlorambucil, Melphalan, dacarbazine, Nitrosoureas, and Temozolomide.
  • Achemotherapeutic agent may be an anthracycline.
  • Non-limiting examples of anthracy clines are Daunorubicin, Doxorubicin, Epirubicin., Idarubicin, Mitoxantrone, and Valrubicin.
  • a chemotherapeutic agent may be a taxane.
  • Non-limiting examples of taxanes are Paclitaxel, Docetaxel, Abraxane, and Taxotere.
  • a chemotherapeutic agent may be ahistone deacetylase inhibitor.
  • Non limiting examples of histone deacetylase inhibitors are Vorinostat and Romidepsin.
  • a chemotherapeutic agent may be a topoisomerase inhibitor.
  • Non-limiting examples of topoisomerase inhibitors are Irinotecan, Topotecan, Etoposide, Teniposide, and Tafluposide.
  • a chemotherapeutic agent may be a kinase inhibitor.
  • Non-limiting examples of kinase inhibitors are Bortezomib, Erlotinib, Gefitinib, Imatinib, Vemurafenib, and Vismodegib.
  • a chemotherapeutic agent may be a nucleotide analog or precursor analog.
  • Non-limiting examples of nucleotide analogs or precursor analogs are Azacitidine, Azathioprine, Capecitabine, Cytarabine, Doxifluridine, Fluorouracil, Gemcitabine, Hydroxyurea, Mercaptopurine, Methotrexate, and Tioguanine.
  • a chemotherapeutic agent may be a platinum based agent.
  • Non-limiting examples of platinum based agents are Carboplatin, Cisplatin, and Oxaliplatin.
  • a chemotherapeutic agent may be a retinoid.
  • Non-limiting examples of retinoids are Tretinoin, Alitretinoin, and Bexarotene.
  • a chemotherapeutic agent may be a vinca alkaloid or derivative.
  • Non-limiting examples of vinca alkaloids or derivatives are Vinblastine, Vincristine, Vindesine, and Vinorelbine.
  • the therapy resistance is resistance to an a chemotherapeutic agent as listed above.
  • the resistance to an a chemotherapeutic agent is resistance to Paclitaxel, Docetaxel, Abraxane, Taxotere, Cabazitaxel, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Estramustine phosphate, Alestramustine, Atrimustine, Cytestrol acetate, Estradiol mustard, Estramustine, Estromustine, ICI-85966, or Phenestrol.
  • the resistance to an a chemotherapeutic agent is resistance to Ocetaxel, Cabazitaxel, Mitoxantrone, Estramustine.
  • the present invention further provides that an additional therapy is administered to the subject, preferably a therapy to which resistance is avoided or reversed by the product as defined herein.
  • the treatment, prevention or amelioration further comprises administering an antiandrogen.
  • the antiandrogen may be selected from the antiandrogens described above.
  • the treatment, prevention or amelioration further comprises administering an antiandrogen selected from Enzalutamide, Flutamide, Nilutamide, Bicalutamide, Topilutamide, Apalutamide, Darolutamide, Cimetidine, ketoconazole, Proxalutamide (GT-0918), or Seviteronel (VT- 464), or Abiraterone.
  • the treatment, prevention or amelioration further comprises administering Enzalutamide.
  • the treatment, prevention or amelioration further comprises administering a PARP inhibitor.
  • the PARP inhibitor may be selected from the PARP inhibitors described above.
  • the treatment, prevention or amelioration further comprises administering a PARP inhibitor selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, Pamiparib (BGB-290), Rucaparib, CEP 9722, or E7016.
  • the treatment, prevention or amelioration further comprises administering Olaparib.
  • the treatment, prevention or amelioration further comprises administering a chemotherapeutic agent.
  • the chemotherapeutic agent may be a chemotherapeutic agent as listed above.
  • the treatment, prevention or amelioration further comprises administering a chemotherapeutic agent selected from: Paclitaxel, Docetaxel, Abraxane, Taxotere, Cabazitaxel, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Estramustine phosphate, Alestramustine, Atrimustine, Cytestrol acetate, Estradiol mustard, Estramustine, Estromustine, ICI- 85966, or Phenestrol.
  • the treatment, prevention or amelioration further comprises administering a chemotherapeutic agent selected from to Ocetaxel, Cabazitaxel, Mitoxantrone, Estramustine.
  • the product as broadly described herein is administered to a subject in case the subject demonstrates therapy resistance.
  • the therapy resistance is androgen deprivation resistance or PARP inhibitor resistance.
  • the product as broadly described herein is adminetered to a subject in case the tumor from the subject has low PDE4D7 expression, as it is envidsioned that in such cases it is very likely that PDE4D7 expression is reduced and that the subject may benefit form the product as broadly described herein.
  • the invention relates to a product for use in the treatment, prevention or amelioration of prostate cancer in a subject in need thereof, wherein the product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7, wherein the treatment, prevention or amelioration comprises providing a sample from the subject and determining the expression level of PDE4D7.
  • the invention relates to a method of treating, preventing or ameliorating prostate cancer in a subject in need thereof, the method comprising:
  • the product is administered to the subject when a low expression level of PDE4D7 is observed in the sample.
  • an alternative treatment (e.g. not the product as defined herein) is administered to the subject when a high expression level of PDE4D7 is observed in the sample.
  • the alternative treatment may for example be a conventional treatment option for prostate cancer.
  • the alternative treatment in patients with localized disease is active surveillance (instead of active treatment).
  • the alternative treatment in patients with non-localized disease is conventional / standard-of-care treatment or treatment de-intensification.
  • the sample is a tumor sample or a tissue sample from the subject, preferably the sample is a tumor sample, more preferably a prostate cancer tumor sample.
  • the tumor sample may be a biopsy or culture obtained from the tumor.
  • the skilled person will be easily able to determine whether an expression level is deemed “high” or “low”, for example by creating a reference library of prostate tumor samples and determine PDE4D7 expression it can easily be determined what the overall range of PDE4D7 specific expression levels are found in tumor samples and which values correspond to a high value (e.g. above the median value) and which values correspond to a low value (e.g. below the median value).
  • a compound for use in the treatment, prevention or amelioration of prostate cancer in a subject in need thereof wherein the compound is an ATR inhibitor or an metabolic inhibitor.
  • the compound is adminstered if:
  • the compound is an ATR inhibitor.
  • the ATR inhibitor is selected from RP-3500, Ceralasertib (AZD6738), AZ20, Elimusertib (BAY-1895344), Elimusertib (BAY-1895344) hydrochloride, SKLB-197, ETP -46464, Berzosertib (VE-822), Dactolisib (BEZ235), VE-821, Torin 2, or CGK 733.
  • the ATR inhibitor is Ceralasertib.
  • the compound is a metabolic inhibitor. Suitable metabolic inhibitors are known to the skilled person and for example described in Lemberg et al., J Clin Invest.
  • the metabolic inhibitor is selected from Methotrexate, Pemetrexed, 6-Mercaptopurine, 6- Thioguanine, Capecitabine, 5 -Fluorouracil, Gemcitabine, Cytarabine, Leflunomide, CB-839, PEG-BCT- 100 (ADIPEG20), AEB-1102, L-Asparaginase, TVB-2640, AG-120 (ivosidenib), IDH305, BAY1436032, FT-2102, AG-221 (enasidenib), AG-881, AZD3965, CPI-613, or Metformin.
  • the metabolic inhibitor is metformin.
  • the invention further provides a kit of parts comprising a product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7 as broadly defined herein. Therefore, in an embodiment the kit comprises a compound or composition wherein the compound or composition induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7.
  • the product is selected from: a gene therapy for inducing PDE4D7 expression; a compound that induces PDE4D7 expression; a compound directly stimulating or promoting phosphodiesterase 4D7 activity; a compound indirectly stimulating or promoting phosphodiesterase 4D7 activity; an inhibitor of a transcriptional inhibitor of PDE4D7; an inhibitor of an inhibitor of phosphodiesterase 4D7 enzymatic activity mRNA encoding a phosphodiesterase 4D7 protein; a phosphodiesterase 4D7 protein; a phosphodiesterase 4D7 activity promoting peptide.
  • the gene therapy is selected from: a viral vector capable of expressing PDE4D7 in a subject or a non-viral vector capable of expressing PDE4D7 in a subject.
  • the product is comprised in a nanoparticle.
  • kit of parts further comprises an antiandrogen and/or a PARP inhibitor and/or a chemotherapeutic agent.
  • antiandrogen is selected from Enzalutamide, Flutamide, Nihitamide, Bicalutamide, Topilutamide, Apalutamide, Darolutamide, Cimetidine, ketoconazole, Proxalutamide (GT-0918), Seviteronel (VT-464), or Abiraterone.
  • the PARP inhibitor is selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, Pamiparib (BGB-290), Rucaparib, CEP 9722, or E7016.
  • the chemotherapeutic agent is selected from Paclitaxel, Docetaxel, Abraxane, Taxotere, Cabazitaxel, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Estramustine phosphate, Alestramustine, Atrimustine, Cytestrol acetate, Estradiol mustard, Estramustine, Estromustine, ICI-85966, or Phenestrol.
  • the invention relates to the ex vivo or in vitro use of a product that induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7as broadly defined herein to increase expression of PDE4D7 and/or activity of phosphodiesterase 4D7 in a cell.
  • any method, use or composition described herein can be implemented with respect to any other method, use or composition described herein.
  • Embodiments discussed in the context of methods, use and/or compositions of the invention may be employed with respect to any other method, use or composition described herein.
  • an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.
  • All cells were cultured in RPMI 1640 medium (GibcoD), supplemented with 10% (v/v) foetal bovine serum, 1% (v/v) L-Glutamine and 1% (v/v) penicillin/streptomycin, and stored within a 37°C incubator with 5% CO2.
  • Table 1 LNCaP FGC cells transduced with lentiviral shRNA for different levels of PDE4D7 expression.
  • LNCaP WT and stable transduced cell lines were transiently transfected with pcDNA3.1-PDE4D7-VSV plasmid DNA construct using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific) according to manufacturer’s protocol. Cells were incubated for 24h post-transfection prior to further experiments.
  • RTCA Real-Time Cell Analysis
  • CI cell index
  • RTCA Real-time cell growth was analysed using the xCELLigence RTCA system (Agilent), which measures real-time cell adhesion across gold microelectrode biosensors on 96 well electronic plates (E-plates) within a 37°C incubator with 5% CO2. Cellular adhesion influences electrical impedance which is converted into the cell index (CI) via RTCA software.
  • Initial background measurements were recorded with 100 pL RPMI 1640 medium, followed by addition of 100 pL LNCaP cell suspension (10,000 cells/wells) for measurement of CI over time. Approximately 24h after seeding, cells were treated with specified concentrations of drugs. Negative controls containing either 0.1% DMSO or untreated cells were also included.
  • CI was transformed to normalised CI via the RTCA software to timepoint of treatment.
  • Cells were lysed in 3T3 lysis buffer (50 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 25 mM HEPES, 2.5 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100; pH 7.5) supplemented with cOmpleteTM EDTA-free protease inhibitor cocktail (Sigma- Aldrich). Lysed cells were rotated end-over- end for 30 minutes at 4°C, then centrifuged for 10 minutes at 13,000 RPM at 4oC. Supernatants were collected and protein concentration determined via Bradford assay.
  • 3T3 lysis buffer 50 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 25 mM HEPES, 2.5 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100; pH 7.5
  • Equal concentrations of protein samples were separated via SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes for western blot. Membranes were blocked in 5% (w/v) milk powder in TBS-T for Ih prior to primary and secondary antibody incubations.
  • anti-PDE4D5 1:5000 Baillie lab
  • anti-PDE4D7 1:500 Baillie lab
  • anti-PDE4D9 1:5000 Baillie lab
  • anti-Pan- PDE4D 1:5000 Baillie lab
  • anti-VSV 1:5000 Abeam, #abl874
  • anti-GAPDH 1:5000 Abeam, #ab8245
  • anti-AR 1:1000 Abeam, #
  • anti-PSMA 1:1000 anti-E-cadherin 1:500
  • anti-TMPRSS2 1:1000 Abeam #abl09131
  • anti-NKX3.1 1:1000 Cell Signalling, #92998
  • anti-PSA 1:1000 Abeam, #ab76113
  • anti-AR-V7 Cell Signalling, #68492
  • anti-GR Cell Signalling, #12041T
  • RNAseq next generation RNA sequencing
  • NovaSeq 6000 S4 system 2x150 bp read length
  • RNAseq reads were mapped to the human reference genome (Ensembl 96) using STAR.
  • Transcript per million (TPM) values were analysed between WT and shRNA transduced LNCaP cell lines and converted into z-scores.
  • RNAseq data was further analysed via iPathwayGuide (Advaita Bio) to calculate the log2 fold change of genes associated with the prostate cancer pathway.
  • LNCaP cells were seeded onto 13mm glass coverslips 24 hours prior to transfection as previously described. Cells were fixed with 4% (v/v) paraformaldehyde for 15 minutes, blocked and permeabilised with 10% donkey serum, 0.5% BSA and 0.2% Triton-X in PBS. Coverslips were incubated overnight with anti-PDE4D7 primary antibody (1:200, Baillie Lab), followed by incubation in Alexa-Fluor 488 Donkey -anti-Goat 1:500 secondary antibody (Thermo Fisher Scientific, #A-11055). Coverslips were mounted onto slides with Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich) and imaged via the ZEISS LSM 880 laser scanning microscope with 63x oil immersion objective.
  • DAPI Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich) and imaged via the ZEISS LSM 880 laser scanning microscope with 63x oil immersion objective.
  • PDE4D7-knockdown LNCaP cell line was generated via shRNA-mediated lentiviral transduction.
  • Pl cells revealed the most significant PDE4D7 mRNA-mediated downregulation via qPCR (Fig. 7) and was chosen alongside the WT and scrambled-shRNA LNCaP cell lines to look into the effects of PDE4D7 expression in prostate cancer.
  • Proof of significant PDE4D7 knockdown at mRNA [Fig. 1 A] and protein level [Figs. IB] in the Pl LNCaP cell line compared to WT was obtained.
  • the reduction in PDE4D7 protein expression was also confirmed using confocal microscopy [Fig. 1C] .
  • RNA seq Analysis of alterations in genomic profiles between WT, scrambled and Pl cells were determined via NGS RNA seq.
  • Top differentially expressed genes [Fig. ID] are shown as z-scores between WT and Pl cells, with many of these genes exhibiting significant downregulation in Pl LNCaPs.
  • Adharaa pathway analysis was performed on RNA seq data, and it identified that the top differentially expressed genes are implicated in PCa-related signalling pathways [Fig. IE], with logFC downregulated most significantly for KLK3, AR, SPINT1, TMPRSS2 and NKX3.1.
  • Fig. IE PCa-related signalling pathways
  • PDE4D7-knockdown in LNCaP cells alters phenotype and confers resistance to enzalutamide Knockdown of PDE4D7 in LNCaP cell line Pl significantly enhanced growth characteristics of the cells.
  • xCELLigence growth assays were also performed on WT, Pl and the scrambled-shRNA knockdown (SC2) LNCaP cell lines.
  • Real-time growth curves showed enhanced growth of Pl cells in comparison to both WT and SC2 [Fig. 2A], correlating with a significant difference in slope analysis. Importantly, no significant difference in growth characteristics were found between the WT and SC2 LNCaP cells.
  • Enzalutamide is an AR antagonist used in the treatment of advanced PCa (Menon and Higano, 2013). Given that significant down-regulation of PDE4D7 expression is a characteristic of androgen-insensitive prostate cancer (Henderson et al., 2014), we wanted to investigate the effects on the growth of WT and PDE4D7-silenced PCa cells following treatment with enzalutamide. To assess optimum treatment concentrations in WT and Pl LNCaP cells, a dose response experiment was performed using increasing drug concentrations and monitored via xCELLigence technology. All concentrations tested resulted in a significant decrease in WT LNCaP cell proliferation (0.1-30 pM), with IpM and above resulting in the least growth [Fig.
  • PDE4D7 -knockdown in LNCaP cells confers resistance to P ARP -inhibition and is rescued upon reintroduction ofPDE4D7
  • Olaparib is an FDA-approved Poly (ADP -ribose) polymerase (PARP) inhibitor that is effective against LNCaP cells (Feiersinger et al., 2018). Ceralasertib is an ataxia-telangiectasia mutated and Rad3-related (ATR) inhibitor (AZD6738), that has been suggested to enhance synergistic effects in combination with olaparib in mCRPC (Lloyd et al., 2020). In light of this, we wanted to determine whether PDE4D7- knockdown affected LNCaP cells response to either of these treatments. Using real-time growth analysis, LNCaP WT proliferation decreased in a dose-dependent manner with olaparib [Fig.
  • PDE4D7-knockdown in LNCaP cells does not affect response to ATR inhibition yet may limit docetaxel treatment response
  • Docetaxel is a DNA-damage inducing chemotherapy drug and we decided to utilise it to discover whether there is a difference between the ability of WT and Pl cells to repair DNA and hence survive upon treatment.
  • docetaxel significantly reduced growth of both WT and PDE4D7- knockdown LNCaPs at all concentrations (Figs. 4A and 4B, respectively], except for lOpM in Pl cells which appeared to have no effect.
  • lOpM was effective in WT but not Pl
  • Pl we investigated how PDE4D7-reintroduction to Pl cells affected this. As with olaparib [Fig. 3D], P1-PDE4D7+/+ cells were more sensitive to docetaxel treatment than Pl [Fig. 4C], suggesting that a paucity of PDE4D7 promotes the ability to repair associated DNA damage and protects against induction of cell death.
  • RNA seq genes identified as differentially expressed between WT and Pl cells are implicated in anti-androgen resistance.
  • Signalling via the glucocorticoid receptor (GR) can bypass AR inhibition in CRPC patients leading to enhanced proliferation [REF]
  • GR expression was upregulated in Pl and confirmed via western blot as significant protein upregulation [Fig. 5A]
  • Real-time growth analysis revealed that neither WT or Pl cell proliferation were reduced compared to DMSO from 0.3-10pM mifepristone treatment [Figs.
  • CUTie cAMP FRET probes have been shown to have many advantages over conventional constructs [REF]
  • CUTie probes targeting the plasmamembrane (AKAP-79) or cytosol (CYTO-pDUAL) were obtained from the Zaccolo lab (REF).
  • Treatment with rolipram induced a downregulation of FRET ratio in the membrane compartment of WT LNCaPs, indicating an increase in cAMP concentration.
  • Metformin is a well-known drug used to treat type-2 diabetes, however its potential in treating numerous cancers has shown promising results [REF], With regards to PCa it remains controversial, where it has shown to enhance overall survival/time to recurrence, however no evidence of it treating/reducing the PCa tumours itself (He et al., 2019). Given that metformin is known to decrease cAMP production (Miller et al., 2013), we decided to test its effects between WT and Pl LNCaPs. Analysis of growth phenotype shows that metformin was successful at significantly reducing growth of WT cells at all concentrations [Fig. 6E], however significant inhibition was only observed in Pl cells at highest concentrations tested [Fig. 6F],
  • ML-R2 is a PDE4 longform activator compound that binds allosterically to the enzyme in order to mimic PKA phosphorylation in the UCR1 domain [REF]
  • PDE4D7 knockdown leads to an enhanced growth phenotype in LNCaPs
  • our results revealed that, whilst there was no effect on proliferation in WT LNCaPs at either concentration [Fig. 6G], ML-R2 addition caused inhibition of cell growth in Pl cells [Fig.
  • MLR2 has no effect on growth of WT LNCaPs, MLR2 attenuates growth of Pl LNCaPs and stable PDE4D7-reintroduction to Pl LNCaPs rescues phenotype of WT LNCaPs on MLR2 sensitivity.
  • P1+4D7 cells were treated with DMSO.
  • A+B) Reintroduction of PDE4D7 to Pl enhances sensitivity to Olaparib, however reintroduction of PDE4D7 alone also reduces growth to same extent.
  • 367 patients managed at two prostate cancer centers in Germany were included into this study.
  • 363 patients had at least one adverse feature in pathology (PSM: positive surgical margins; SVI: seminal vesicle invasion; EPE: extra-prostatic extension; LNI: lymph node invasion; Gleason 4 component) and/or were classified into post-treatment intermediate or high risk of disease progression according to the clinical risk metrics CAPRA-S (24) and/or the EAU-BCR risk model (25) while 4 patients demonstrated a post-operatively rising PSA in the absence of any of the stated adverse features; all 367 patients experienced biochemical recurrence during post-treatment follow-up and were scheduled to salvage radiation therapy (SRT) of the pelvic bed with a total administered radiation dose between SO- 72 Gy over multiple fractions.
  • SRT salvage radiation therapy
  • Two small biopsy punches (-1x2 mm) of a representative resected tumor area of 367 patients operated on between 1994-2011 were collected from the tumors index lesion.
  • the local Institutional Review Boards approved the collection of patient tissue for clinical research.
  • LNCaP clone FGC (ATCC® CRL1740TM) were cultured in RPMI 1640medium (GibcoTM), supplemented with 10% (v/v) foetal bovine serum, 1% (v/v) L-Glutamine and 1% (v/v) penicillin/streptomycin, and stored within a 37°C incubator with 5% CO2.
  • the transfer plasmid of the lentivirus transfection system was designed to stably express and shRNA RNA hairpin targeting the first coding exon of the PDE4D7 transcript.
  • a scrambled nucleotide sequence of the PDE4D7 targeting shRNA was cloned into the transfer plasmid of the lentivirus.
  • LNCaP FCG wildtype WT
  • LNCaP transfected with scrambled shRNA control clone SC2
  • LNCaP transfected with PDE4D7 targeting shRNA clones Pl and w5.2
  • LNCaP clone Pl transfected with Tet-On inducible PDE4D7 vector clone P1-TET4D7.
  • tumour heterogeneity To account for potential tumour heterogeneity the two tissue punches of the RP cohort were combined before nucleic acid extraction. A potential difference in tumour cellularity was addressed by normalisation of the PDE4D transcript expression to four reference genes. All used molecular laboratory methods including RNA extraction, quantitative real-time PCR (RT-qPCR), oligonucleotide primers/probes and statistical analysis were as previously described (21).
  • the post-surgical CAPRA-S risk score and its corresponding low (1), intermediate (2), high-risk (3) categories were calculated as described earlier (24).
  • the EAU-BCR score and its two categories of low and high post-surgical risk of disease progression was determined as published (25).
  • Two cut-offs for the pPDE4D7 score were defined by AUROC (Area under the ROC curve) analysis with 5-year prostate cancer specific death after start of SRT as the dependent variable and the pPDE4D7 score as the independent variable.
  • One cut-off (pPDE4D7>0.2) was defined as the point in the AUROC with maximum sensitivity and specificity.
  • the cut-off was defined as max sensitivity (pPDE4D7>0.87).
  • RNA sequencing was perfomred as described in Example 1.
  • GSEA Gene Set Enrichment Analysis
  • GSEA Gene Set Enrichment Analysis
  • the concentration was determined by performing qPCR on the samples using the 2X KAPA HiFi HotStart ReadyMix (Kapa Biosystems) as mastermix and a dilution of PhiX Sequencing Control v3 as standard. High coverage sequencing was performed on the Illumina Novaseq 6000 system with the S4 flowcell and PE150 configuration.
  • Plasmid DNA transfection, eCELLigence Real-time Cell analysis, Western Blotting, and Immunofluorescence were performed as described in Example 1.
  • the software package MedCalc MedCalc Software v2.20 BVBA, Ostend, Belgium
  • values are presented as mean ⁇ standard error of mean (S.E.M) or standard deviation (SD) for N>3, unless otherwise stated.
  • the PDE4D7 score is associated with survival outcomes in high-risk prostate cancer patients
  • Multivariate Cox regression analysis was conducted to assess the association between the PDE4D7 score and prostate cancer-specific mortality (PCSM) after SRT initiation, adjusting for pathological prognostic variables (i.e., ISUP Gleason grade group, pathological pT stage, status of extra-prostate extension and surgical margins, seminal vesicle involvement, and lymph node invasion).
  • PCSM prostate cancer-specific mortality
  • the survival rate in the low-risk group was greater than 90%, while it was approximately 72% in the high-risk group.
  • the CAPRA-S model did not show significant stratification between risk groups.
  • ROC receiver operating characteristic
  • AUC area under the curve
  • PDE4D7 knockdown is associated with loss of androgen signaling, enrichment of the mesenchymal epithelial transition, and neuroendocrine prostate cancer.
  • LNCaP clone FCG
  • LNCaP Pl cells exhibited the most significant downregulation of PDE4D7 mRNA expression, as confirmed by RT-qPCR.
  • Western blot analysis and confocal microscopy further confirmed the reduction in PDE4D7 protein expression in LNCaP Pl cells compared to the wildtype LNCaP and a scrambled shRNA control (LCNaP_SC2) ( Figures 1A, IB, and 1C).
  • the most down-regulated genes in the two PDE4D7 knock-down cell lines were the known AR-regulated kallikrein-related peptidases KLK2 and KLK3, the transmembrane serine protease 2 (TMPRSS2), and the transcription factor NK3 homeobox-1 (NKX3-1). Also, the AR itself - although not part of this hallmark AR response pathway - is strongly downregulated in the PDE4D7 knock-down cell line Pl ( Figure IF).
  • the described findings indicate that the selective depletion of the PDE4D7 transcript expression leads to a cellular phenotype which represents androgen-insensitive proliferation.
  • NED in prostate cancer is a hallmark of aggressive, AR-independent, and treatment-resistant disease. It can occur in both primary and metastatic prostate cancer. NED is characterized by decreased expression of the AR and increased expression of neuroendocrine markers.
  • PDE4D7-knockdown in LNCaP cells alters phenotype and confers resistance to AR antagonist Knockdown of PDE4D7 in LNCaP cell line Pl resulted in increased growth characteristics of the cells, as confirmed by real-time growth assays comparing Pl PDE4D7-knockdown cells to WT and SC2 LNCaP cells ( Figure 2A). Importantly, no significant difference in growth characteristics were found between the WT and SC2 LNCaP cells.
  • Enzalutamide an AR antagonist used to treat advanced PCa, was utilized to investigate the effects on growth of PDE4D7-silenced PCa cells.
  • Dose-response experiments using real-time proliferation technology showed a significant decrease in WT LNCaP cell proliferation at all concentrations tested (0.1-30 pM), with the most profound effects at IpM and above ( Figure 2B).
  • Pl LNCaP cells showed minimal reductions in proliferation upon treatment with enzalutamide and even exhibited increased growth at higher concentrations (Figure 2C).
  • Re-expression of PDE4D7 in LNCaP Pl cells via a Tet-On inducible vector rescued the growth phenotype and restored sensitivity to enzalutamide ( Figure 15A, 15B). This data strongly highlights that low PDE4D7 expression in PCa cells confers resistance to anti-androgens such as enzalutamide.
  • MR-L2 a PDE4 longform activator that binds allosterically to the PDE4D enzyme in order to mimic PKA phosphorylation in the UCR1 domain, was tested to rescue the enhanced growth phenotype caused by PDE4D7 knockdown in LNCaPs.
  • Results showed inhibition of growth in Pl cells, but no effect on proliferation in WT LNCaPs ( Figure 2E, 2F). This data suggests that activation of PDE4 in PDE4D7- knocked down LNCaPs has an inhibitory effect on their proliferative potential and highlights again the importance of PDE4D7 in driving PCa.
  • PDE4D7 specific knockdown in LNCaPs is associated with expression changes in DNA damage repair genes
  • PDE4D7 knockdown in LNCaPs confers resistance to PARP-inhibition and is rescued upon reintroduction of PDE4D7
  • Olaparib is an FDA-approved PARPi that is effective against LNCaP cells.
  • Ceralasertib is an ataxiatelangiectasia mutated and Rad3-related (ATR) inhibitor (AZD6738), which has been suggested to enhance synergistic effects in combination with olaparib in metastatic CRPC.
  • ATR ataxiatelangiectasia mutated and Rad3-related
  • AZD6738 ataxiatelangiectasia mutated and Rad3-related
  • PDE4D7 knockdown affects LNCaP cells response to either of these treatments.
  • LNCaP WT proliferation decreased in a dose-dependent manner with olaparib ( Figure 3A), whereas the effect on PDE4D7-deficient LNCaPs was minimal (Figure 3B).
  • P1-PDE4D7 111811 cells were resensitised to lOpM olaparib treatment (Figure 3E), as was previously apparent in the WT LNCaP cells, with a statistically significant downregulation in growth recorded when compared to both olaparib-treated Pl cells, and DMSO-treated P1-PDE4D7 +/+ cells. This data re-confirms the role of PDE4D7 is blunting of PCa cell growth and promoting susceptibility to clinically relevant PCa therapeutics.
  • PDE4D7-knockdown in LNCaP cells does not affect response to ATR inhibition yet may limit docetaxel treatment response
  • Docetaxel is a DNA-damage inducing cytotoxic drug which is clinically used in advanced prostate cancer treatment. Compared to DMSO, docetaxel significantly reduced growth of both WT and PDE4D7- knockdown LNCaPs at all concentrations, except for lOpM in Pl cells which appeared to have no effect. Given that lOpM was effective in WT but not Pl, we investigated how PDE4D7-reintroduction to Pl cells affected this. As with olaparib, P1-PDE4D7 +/+ cells were more sensitive to docetaxel treatment than Pl, suggesting that a paucity of PDE4D7 promotes the ability to repair associated DNA damage and protects against induction of cell death. This is in line with the increased expression of several key DNA repair genes in PDE4D7-knockdown LNCaPs.
  • Ceralasertib and metformin appear to inhibit LNCaP cell growth irrespective of PDE4D7 expression status (wt vs Pl, see Examples 1 and 3) Ceralasertib or metformin may provide an alternative treatment option for prostate cancer patients with resistance to antiandrogens (such as Enzalutatmide) or PARP inhibitors (such as Olaparib), where the tumors have low expression of PDE4D7.
  • antiandrogens such as Enzalutatmide
  • PARP inhibitors such as Olaparib
  • RP radical prostatectomy
  • Post-surgical pathology is given (Note: extracapsular extension was derived from pathology stage information).
  • the outcome category illustrates the cumulative events in terms of recurrence to clinical metastases (CR), salvage radiation therapy (SRT) or androgen deprivation therapy (ADT) after surgery.
  • CR clinical metastases
  • SRT salvage radiation therapy
  • ADT androgen deprivation therapy
  • the PDE4D7 Score and the ISUP Gleason grade group were used as continuous variables; the pathology stage pT was used as categorical variable with pT2 as reference.
  • the Hazard Ratio (HR), 95% confidence interval (CI) of the HR, and the logrank p-value for each survival analysis are indicated.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to a product for use in the treatment, prevention or amelioration of prostate cancer in a subject in need thereof, wherein the product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7. The product may be a direct or indirect activator of PDE4D7 activity or a product for expressing or promoting expression of PDE4D7 in a subject. The invention further defines a kit of parts and an in vitro or ex vivo use of the product.

Description

TREATMENT OF PROSTATE CANCER BY PROMOTING PDE4D7
FIELD OF THE INVENTION
The invention relates to a product for use in the treatment, prevention or amelioration of prostate cancer by reducing therapy resistance in a subject in need thereof. In particular the product is used to induce the expression of PDE4D7 or promote activity of phosphodiesterase 4D7.
BACKGROUND OF THE INVENTION
Cancer is a class of diseases in which a group of cells display uncontrolled growth, invasion and sometimes metastasis. These three malignant properties of cancers differentiate them from benign tumors, which are self-limited, do not invade or metastasize. Among men, the three most commonly diagnosed cancers are prostate, lung and colorectal cancer in developed countries. Particularly prostate cancer is the most common malignancy in European males. In 2002 in Europe, an estimated 225,000 men were newly diagnosed with prostate cancer and about 83,000 died from this disease.
Prostate cancer, for example, is traditionally diagnosed via the serum level of prostatespecific antigen (PSA). However, PSA is not prostate cancer-specific and can be raised in other circumstances, leading to a large number of false-positive s (cancer is not found in around 70% of men with raised PSA levels who undergo biopsy). Furthermore, there will be an unpredictable number of false-negatives who later develop prostate cancer in the presence of a "normal" PSA test.
In patients who undergo treatment, the most important clinical prognostic indicators of disease outcome are the stage, pretherapy PSA level, and Gleason score. The higher the grade and the stage, the poorer the prognosis. Nomograms can be used to calculate the estimated risk of the individual patient. The predictions are based on the experience of large groups of patients. [250] A complicating factor is that the majority of patients have multiple independent tumor foci upon diagnosis, and these foci have independent genetic changes and molecular features. Because of this extensive inter-focal heterogeneity, it is a risk that the prognostication is set based on the wrong tumor focus.
Androgen ablation therapy causes remission in 80-90% of patients undergoing therapy, resulting in a median progression-free survival of 12 to 33 months. After remission, an androgenindependent phenotype typically emerges, wherein the median overall survival is 23-37 months from the time of initiation of androgen ablation therapy. How androgen-independence is established and how it reestablishes progression is unclear. Androgen ablation therapy may for example include taking antiandrogen drugs.
US 2012/129788 Al relates to the 4D7 isoform of PDE and teaches that tumors have increased PDE4D7 expression, and this can be used for diagnosis purposes. EP3434787 Al teaches that expression of PDE4D7 is reduced in hormone resistance and diagnosis using PDE4D7 expression levels.
EP3303618 Al relates to methods for determining gene expression signatures for diagnosing patients with prostate cancer, e.g., to establish a prognosis for such patients, for determining the predisposition of said patient to develop aggressive or indolent disease, for example after primary treatment, and/or for identification and stratification of patients for available therapies.
Henderson et al. (BRITISH JOURNAL OF CANCER, vol. 110, no. 5, 11 February 2014 (2014-02-11), pages 1278-1287) describes that PDE4D7 is downregulated in androgen-independent prostate cancer cells.
Boettcher et al. (ONCOTARGET, vol. 7, no. 43, 25 October 2016 (2016-10-25), pages 70669-70684) describes that PDE4D isoform composition is deregulated in primary prostate cancer and indicative for disease progression and development of distant metastases.
Therefore there is a constant need for new and improved methods for treatment of cancers such as prostate cancer. Moreover, methods for reducing or preventing resistance to treatments such as antiandrogen drugs or PARP inhibitors.
These drawbacks, among others, are overcome by the products and methods as outlined in the appended claims.
SUMMARY OF THE INVENTION
In a first aspect the invention relates to a product for use in the treatment, prevention or amelioration of prostate cancer by reducing therapy resistance in a subject in need thereof, wherein the product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7 and is selected from: a polynucleotide encoding a peptide having a sequence according to SEQ ID NO: 2 or a variant thereof, wherein the variant is a polynucleotide encoding a peptide having a sequence with at least 90% sequence homology with SEQ ID NO: 2 and wherein the peptide has phosphodiesterase activity; or a polynucleotide comprising a nucleotide sequence as defined in SEQ ID NO: 1 or a variant thereof, wherein the variant is a polynucleotide comprising a nucleotide sequence with at least 90% sequence homology to SEQ ID NO: 1 and wherein the polynucleotide encodes a peptide that has phosphodiesterase activity; or a peptide comprising a peptide sequence as defined in SEQ ID NO: 2 or a variant thereof, wherein the variant is a peptide comprising a peptide sequence with at least 90% sequence homology with SEQ ID NO: 2 and wherein the peptide has phosphodiesterase activity; or a peptide comprising a peptide sequence encoded by a polynucleotide comprising a nucleotide sequence as defined in SEQ ID NO: 1, or a variant thereof, wherein the variant is peptide comprising a peptide sequence encoded by a polynucleotide comprising a nucleotide sequence with at least 90% sequence homology to SEQ ID NO: 1 and wherein the polynucleotide encodes a peptide that has phosphodiesterase activity; or a compound as defined in formula I
Figure imgf000005_0001
Formula I wherein
Ri is H, (Ci-4)alkyl or (Ci-4)alkyloxy;
R2 and Re are independently selected from H and;
Rs, R4 and R5 are independently selected from H, halogen, CN, (Ci-4)alkyl and (Ci-4)alkyloxy, the (Ci- 4)alkyl and (Ci-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
R7, Rs, and Rio are independently selected from H and F;
Rs is selected from (Ci-4)alkyl, (Ci-4)alkyloxy, CN and halogen, the (Ci-4)alkyl and (Ci-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros; or a peptide having a sequence comprising or consisting a sequence selected from SEQ ID Nos: 24, 25, 26 or 27, or a polynucleotide encoding a peptide having a sequence comprising or consisting a sequence selected from SEQ ID Nos: 24, 25, 26 or 27.
In a second aspect the invention relates to a compound for use in the treatment, prevention or amelioration of prostate cancer in a subject in need thereof, wherein the compound is an ATR inhibitor or an metabolic inhibitor, the use comprising administering the compound if: high expression of PDE4D7 is observed in a tumor; low expression of PDE4D7 is observed but PDE4D7 activity cannot be sufficiently increased to re-sensitize tumors to treatment with inhibitors; or in metastatic tumors.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1: Generation of PDE4D7-knockdown LNCaP cell lines and differences in PCa- related genes. A) PDE4D7 mRNA expression via qRT-PCR. Fold change relative to LNCaP WT (2- AACt) for shRNA-scrambled (SC2) and shRNA-PDE4D7 knock-down (Pl) B) PDE4D7 protein expression in WT and shRNA-PDE4D7 knock-down (Pl) LNCaPs. C) Immunocytochemistry staining of PDE4D7 protein expression (yellow) in WT and Pl LNCaP. D) Heatmap of z-scores of top differentially expressed genes between WT and Pl LNCaPs via RNAseq. E) Log fold change (FC) of top altered genes in PCa-related pathways. F) Western blot and quantification of protein expression of PCa-related RNA- seq-identified genes in WT and Pl LNCaPs. Error bars represent mean +/- SEM, N=3, * p<0.05, **** pO.OOOL
Fig. 2: PDE4D7 knockdown in LNCaP cells enhances growth and confers resistance to enzalutamide. A) Real-time proliferation of LNCaP WT, shRNA-scrambled (SC2) and shRNA-PDE4D7 knock-down (Pl) cells. Slope analysis via linear regression. B & C Real-time analysis of LNCaP WT (B) and shRNA-PDE4D7 knock-down (Pl) cells (C) with enzalutamide (OpM - 30pM) or 0.1% DMSO. D) AR-V7 protein expression western blot and quantification in WT and Pl LNCaPs. E) Cyclin Bl and F) Cyclin DI protein expression western blot and quantification in WT and Pl LNCaPs. Error bars represent mean +/- SEM, N=3, ** p<0.01, **** pO.OOOl).
Fig. 3: PDE4D7-knockdown in LNCaP cells enhances resistance to olaparib. A & B) Real-time proliferation of LNCaP WT (A) and shRNA-PDE4D7 knock-down (Pl) cells following treatment with olaparib (OpM - 30pM) or 0.1% DMSO. Normalised cell index to timepoint of treatment. Slope analysis via linear regression between 0-30h. C) Protein expression of PARP and cleaved PARP (cP ARP) western blot and quantification between WT and Pl LNCaPs. D) Real-time proliferation of Pl and LNCaP P1-PDE4D7+/+ transient re-expression cells in response to 0.1% DMSO (D) or lOpM Olaparib (E) or 10 pM Docetaxel (F). Normalised cell index to timepoint of treatment. Slope analysis between 0-48h post-treatment. Error bars represent mean ± SEM, N=3, * p<0.05, ** p<0.01, *** pO.OOl, **** pO.OOOL
Fig. 4: DNA damage induction and repair agents affect both WT and PDE4D7- knockdown LNCaPs (Pl). Real-time growth analysis of docetaxel (DMSO: control (0.1%); DOC 5: 5 pM docetaxel; DOC 7.5: 7.5 pM docetaxel; DOC 10: 10 pM docetaxel; DOC 25: 25 pM docetaxel; treatment on WT (A) or Pl (B) LNCaPs, or Pl and (C) P1-PDE4D7+/+ (after transient PDE4D7 reexpression) LNCaPs including the slope analysis 0-48h since treatment. D & E) Real-time proliferation of LNCaP WT (D) and Pl (E) cells with ceralasertib (OpM - 30pM). Slope analysis 0-30h post-treatment. Normalised cell index to timepoint of treatment. Error bars represent mean +/- SEM, N=3, * p<0.05, ** pO.Ol, *** pO.OOl, **** pO.OOOl.
Fig. 5: PDE4D7-knockdown alters LNCaP response to various compounds. A) Glucocorticoid receptor (GR) protein expression western blot and quantification between WT and PDE4D7-knock-down Pl LNCaPs. B & C) Real-time proliferation of WT (B) and Pl (C) LNCaPs upon treatment with 0-30pM mifepristone or 0.1% DMSO (control) including slope analysis 0-48h post- treatment. D & E) Real-time proliferation of WT (D) and PDE4D7-knock-down Pl (E) LNCaPs upon treatment with l-10pM mebendazole or 0.1% DMSO including slope analysis 0-48h post-treatment. Error bars represent mean +/- SEM, N=3, * p<0.05, ** p<0.01, *** p<0.001, **** pO.OOOl.
Fig 6: A & B) Real-time growth analysis of WT (A) and PDE4D7-knock-down Pl (B) cells upon treatment with metformin (0-25 pM) including slope analysis from 0-48h post-treatment. C & D) Real-time growth analysis of WT (C) and PDE4D7-knock-down Pl (D) cells upon treatment with PDE4 activator ML-R2 (l-10pM) including slope analysis from 0-48 post-treatment. Error bars represent mean +/- SEM, N=3, ** pO.Ol, *** pO.OOl, **** p<0.0001.
Fig. 7: Difference in PDE4D isoform mRNA expression between knockdown LNCaP cell lines. RT-qPCR of various PDE4D knockdown LNCaP cell lines shows fold changes in gene expression relative to WT (2-AACt) for PDE4D5 (A), PDE4D7 (B) and PDE4D9 (C.) One-way ANOVA was performed on AACt values. Error bars represent mean ± SEM for N=3 (* = pO.05, ** = pO.Ol, *** = pO.OOl).
Fig 8: RNA-seq identified genomic alterations between WT and scrambled-shRNA (SC2) LNCaPs. Western blot and quantification of protein expression of PCa-related RNA-seq genes in WT and SC2 LNCaPs (N=l).
Fig. 9: Re-expression of PDE4D7 reduces growth. (A) Real-time growth analysis of PDE4D7-knowdown Pl and PDE4D7-knowdown with stable re-introduction of PDE4D7 P1-4D7+ cells. (B) Slope analysis from 0-72 post-onset. (C) Western blot of protein expression of PDE4D7 isoform in P1-4D7+ and Pl cells. Error bars represent mean ± SEM for N=3, ** p<0.01, *** pO.OOl, **** pO.OOOL
Fig. 10: Testing of the PDE4 activating compound MR-L2 on the real-time proliferation of prostate cancer cells; (i) real-time proliferation; (ii) slope analysis of real-time proliferation. A) WT DMSO: LNCaP wild-type DMSO control (0.1%); WT ML-R2 1: LNCaP wild-type with 1 pM ML-R2; WT ML-R2 10: LNCaP wild-type with 10 pM ML-R2; B) P1 DMSO: LNCaP PDE4D7 knockdown cell line Pl DMSO control (0.1%); P1 ML-R2 1: LNCaP PDE4D7 knockdown cell line Pl with 1 pM ML-R2; P1 ML-R2 10: LNCaP PDE4D7 knockdown cell line Pl with 10 pM ML-R2; C) P14D7+/+ DMSO: LNCaP PDE4D7 knockdown cell line Pl with transient re-expression of PDE4D7 DMSO control (0.1%); P14D7+/+ DMSO: LNCaP PDE4D7 knockdown cell line Pl with transient reexpression of PDE4D7 and with ML-R2 (10 pM). Error bars represent mean ± SEM for N=3, ** pO.Ol, *** pO.OOl, **** pO.OOOl.
Fig. 11: Testing of the PARP inhibitor olaparib on the real-time proliferation of prostate cancer cells. A) P1 OLA: LNCaP PDE4D7 knockdown cell line Pl with 10 pM olaparib; P1+4D7 OLA: LNCaP PDE4D7 knockdown cell line Pl with transient re-expression of PDE4D7 and with 10 pM olaparib; P1+4D7 DMSO: LNCaP PDE4D7 knockdown cell line Pl with transient re-expression of PDE4D7 DMSO control (0.1%); B) slope analysis of real-time proliferation. Error bars represent mean ± SEM for N=3, ** p<0.01, *** p<0.001, **** pO.OOOl. Fig. 12: Ratio of PARP and cleaved PARP (PARPc) by western blot analysis in LNCaP WT and LNCaP PDE4D7 knockdown cell line Pl upon incubation with 0.1% DMSO (control), 10 pM Olaparib, and 10 nM docetaxel including quantitation of western blot bands normalised to DMSO.
Fig. 13: Survival analysis of the PDE4D7 Score to prostate cancer specific mortality (PCSM) after start of salvage radiation therapy (SRT). Two cut-offs for the pPDE4D7 score were defined by AUROC (Area under the ROC curve) analysis with 5-year prostate cancer specific death after start of SRT as the dependent variable and the pPDE4D7 score as the independent variable. One cut-off (pPDE4D7>0.2) was defined as the point in the AUROC with maximum sensitivity and specificity. The cut-off was defined as max sensitivity (pPDE4D7>0.87). Consequently, the pPDE4D7 score classes stratified patients into three sub-cohorts (pPDE4D7 scores >0.87: ‘high’; pPDE4D7 scores >0.2 and <=0.87: ‘intermediate’, and pPDE4D7 scores <=0.2: ‘low’). (A) Kaplan Meier survival analysis of the time to prostate cancer specific death after start of SRT in N=348 clinical high risk prostate cancer patient cohort. Patient groups are stratified according to their PDE4D7 Score with high scores being associated with less risk of death due to prostate cancer compared to low PDE4D7 scores. (B) Kaplan Meier survival analysis of the time to death due to all causes after start of SRT in N=348 clinical high risk prostate cancer patient cohort. (C) Kaplan Meier survival analysis of the time to prostate cancer specific death after start of SRT in N=348 clinical high risk prostate cancer patient cohort. Patient groups are stratified according to the EAU-BCR Risk Score which stratifies patients into a low vs. a high-risk class. (D) Kaplan Meier survival analysis post-SRT of the time to prostate cancer specific death after start of ADT in a N=179 prostate cancer patient cohort. (E) Kaplan Meier survival analysis post-SRT of the time to death due to all causes after start of ADT in a N=179 prostate cancer patient cohort. Logrank p-values are indicated.
Fig. 14: Western blot and quantification of AR-V7 expression in WT and Pl LNCaPs. Mean +/- SEM, N=3, * p<0.05, **** pO.OOOl.
Fig. 15: PDE4D7 knockdown enhances growth and confers enzalutamide resistance, which is rescued upon PDE4D7 re-expression. (A) Real-time growth analysis of P1-TET4D7 +- doxycycline induction of PDE4D7 alone or (B) in response to enzalutamide lOpM.
Fig. 16: Expression boxplot of the BRCA2 gene in PDE4D7 expression classes in human clinical patient samples. The expression of each gene is provided after TPM calculation based on the RNAseq count data. The PDE4D7 classes represent different categories of PDE4D7 expression based on the PDE4D7 score (see references) where PDE4D7_classl (most left-hand box) represents the lowest PDE4D7 scores (i.e., lowest PDE4D7 expression) while PDE4D7_class4 (most right-hand box) represents the highest PDE4D7 scores (i.e., highest PDE4D7 expression). The number of patients per class are: PDE4D7_classl (N=13); PDE4D7_class2 (N=134); PDE4D7_class3 (N=301); PDE4D7_class4 (N=85). The median of the expression per group is indicated by the bar within each box. The red cross represents the mean expression value per group. The circles represent outlier expression values. The p- values were calculated by use of ANOVA Kruskal-Wallis test and represent a significant change in expression over the four PDE4D7 classes.
Fig. 17: (A & B) Real-time proliferation of LNCaP WT (E) and Pl (F) cells with ceralasertib (OpM - 30pM). Slope analysis 0-30h post-treatment. Normalised cell index to timepoint of treatment. Mean ± SEM, N=3, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig. 18: Influence of PDE4D7 expression on cAMP dynamics within LNCaPs.
Analysis of FRET ratio (YFP/CFP) at cytosol of WT (A) or Pl (C) LNCaPs, or at membrane of WT (B) or Pl (D) LNCaPs upon treatment with rolipram or combined treatment of forskolin + IBMX. (E & F) Real-time growth analysis of WT (G) and Pl (H) cells upon treatment with PDE4 activator MR-L2 (1- lOpM). Slope analysis from 0-48 post-treatment. Mean +/- SEM, N=3, ** p<0.01, *** p<0.001.
Definitions
Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense.
Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given.
As used in this specification and in the appended claims, the singular forms of “a” and “an” also include the respective plurals unless the context clearly dictates otherwise.
In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%.
“About” and “approximately" : these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
"Antagonist" and "inhibitor": These terms are used interchangeably, and they refer to a compound or agent having the ability to reduce or inhibit a biological function of a target protein or polypeptide, such as by reducing or inhibiting the activity or expression of the target protein or polypeptide. Accordingly, the terms "antagonist" and "inhibitor" are defined in the context of the biological role of the target protein or polypeptide. An inhibitor need not completely abrogate the biological function of a target protein or polypeptide, and in some embodiments reduces the activity by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. While some antagonists herein specifically interact with (e.g., bind to) the target, compounds that inhibit a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target protein or polypeptide are also specifically included within this definition. Non-limiting examples of biological activity inhibited by an antagonist include those associated with the development, growth, or spread of a tumor, or an undesired immune response as manifested in autoimmune disease.
"Anti-cancer effect" : This refers to the effect a therapeutic agent has on cancer, e.g., a decrease in growth, viability, or both of a cancer cell. The IC50 of cancer cells can be used as a measure the anti-cancer effect. IC50 refers to a measure of the effectiveness of a therapeutic agent in inhibiting cancer cells by 50%.
“Alleviating cancer”: The term, in the context of specific cancers and/or their pathologies, refers to degrading a tumor, for example, breaking down the structural integrity or connective tissue of a tumor, such that the tumor size is reduced when compared to the tumor size before treatment. “Alleviating” metastasis of cancer includes reducing the rate at which the cancer spreads to other organs.
“Compositions”, “products” or “combinations”: These encompass those compositions suitable for various routes of administration, including, but not limited to, intravenous, subcutaneous, intradermal, subdermal, intranodal, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral or mucosal application. The compositions, formulations, and products according to the disclosure invention normally comprise the drugs/compound/inhibitor (alone or in combination) and one or more suitable pharmaceutically acceptable excipients or carriers.
“Combination therapy”, or "in combination with": These term refers to the use of more than one compound or agent to treat a particular disorder or condition. For example, Compound 1 may be administered in combination with at least one additional therapeutic agent. By "in combination with," it is not intended to imply that the other therapy and Compound 1 must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of this disclosure. Compound 1 can be administered concurrently with, prior to (e.g. , 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks before), or subsequent to (e.g. , 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks after), one or more other additional agents. In general, each therapeutic agent will be administered at a dose and/or on a time schedule determined for that particular agent. The other therapeutic agent can be administered with Compound 1 herein in a single composition or separately in a different composition. Higher combinations, e.g., triple therapy, are also contemplated herein.
It is to be understood that the term “comprising” is not limiting. For the purposes of the present invention the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only. Furthermore, the terms “first”, “second”, “third” or “(a)”, “(b)”, “(c)”, “(d)” etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
In case the terms “first”, “second”, “third” or “(a)”, “(b)”, “(c)”, “(d)” etc. relate to steps of a method or use there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.
As used herein, the term "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ... , etc. As used herein, the term "at most" a particular value means that particular value or less. For example, "at most 5" is understood to be the same as "5 or less" i.e., 5, 4, 3, ... .-10, -11, etc.
As used herein, the word “comprise” or variations thereof such as “comprises” or “comprising” will be understood to include a stated element, integer or step, or group of elements, integers or steps, but not to exclude any other element, integer or steps, or groups of elements, integers or steps. The verb “comprising” includes the verbs “essentially consisting of’ and “consisting of’.
As used herein, the term “conventional techniques” refers to a situation wherein the methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.
As used herein, the term “identity" refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (Computational Molecular Biology, Lesk, A. M., ED., Oxford University Press, New York, 1988; Biocomputing: Informatics And Genome Projects, Smith, D. W., ED., Academic Press, New York, 1993; Computer Analysis Of Sequence Data, Part I, Griffin, A. M., And Griffin, H. G., EDS., Humana Press, New Jersey, 1994; Sequence Analysis In Molecular Biology, Von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer; Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two nucleotide sequences or amino acid sequences, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide To Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., Siam J. Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403).
As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a certain sequence, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference amino acid sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted and/or substituted with another nucleotide, and/or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID NO: X is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: X. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As used herein, the term “in vitro” refers to experimentation or measurements conducted using components of an organism that have been isolated from their natural conditions.
As used herein, the term “ex vivo” refers to experimentation or measurements done in or on tissue from an organism in an external environment with minimal alteration of natural condition. As used herein, the term "nucleic acid", “nucleic acid molecule” and “polynucleotide” is intended to include DNA molecules and RNA molecules. A nucleic acid (molecule) may be singlestranded or double-stranded, but preferably is double-stranded DNA.
As used herein, the terms “sequence” when referring to nucleotides, or “nucleic acid sequence”, “nucleotide sequence” or “polynucleotide sequence” refer to the order of nucleotides of, or within, a nucleic acid and/or polynucleotide. Within the context of the current invention a first nucleic acid sequence may be comprised within or overlap with a further nucleic acid sequence.
As used herein, the term “subject” or “individual” or “animal” or “patient” or “mammal,” used interchangeably, refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo-, sports-, or pet-animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, bears, and so on. As defined herein a subject may be alive or dead. Samples can be taken from a subject post-mortem, i.e. after death, and/or samples can be taken from a living subject.
As used herein, terms "treatment", "treating", "palliating", “alleviating” or "ameliorating", used interchangeably, refer to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit. By therapeutic benefit is meant eradication or amelioration or reduction (or delay) of progress of the underlying disease being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration or reduction (or delay) of progress of one or more of the physiological symptoms associated with the underlying disease such that an improvement or slowing down or reduction of decline is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disease.
DETAILED DESCRIPTION OF EMBODIMENTS
The section headings as used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
A portion of this invention contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent invention, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.
Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention relates, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition as provided herein. The preferred materials and methods are described herein, although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.
The activation of G-protein coupled receptors and subsequent activation of adenyl cyclase catalyzes the conversion of ATP to cAMP, a second messenger involved in the regulation of numerous cellular processes. Phosphodiesterases (PDEs) are a superfamily of enzymes which play the fundamental role of hydrolysing cAMP, leading to its degradation and termination of cell signal transduction. These enzymes define and regulate intracellular cAMP gradients around specific signalling complexes, with each specific isoform having unique functional roles.
Of particular interest is the PDE4 family of enzymes, which is made up of over 25 different isoforms, many of which have important, non-redundant functions. Often, the function of a particular PDE4 isoform is conferred by its unique N-terminal, which acts as a “postcode” to anchor PDE4 enzymes to discrete intracellular domains where they sculpt signal-specific cAMP gradients. PDE4s also contain a catalytic unit and regulatory domains termed “upstream conserved regions one and two” (UCR1/2) which are highly conserved throughout the isoforms. All long-form PDE4s, such as for example PDE4D7, contain UCR1, which contains a PKA motif that becomes phosphorylated during conditions of raised cAMP. Such an action serves to activate PDE4 and rapidly reduce the local concentration of cAMP. This feedback loop underpins the transient nature of cAMP signals and ensures a rapid but fleeting response to activation of Gas-coupled receptors.
Deregulated cAMP signalling is associated with various diseases including cancer, with the PDE4D sub-family of particular interest in prostate cancer (PCa). PDE4D7, a long PDE4D isoform variant, localises to the sub-plasma membrane in PCa cells and its expression inversely correlates with disease progression. Specifically, PDE4D7 mRNA is significantly downregulated from an androgen sensitive (AS) to androgen insensitive (Al) disease phenotype, implicating PDE4D7 in PCa development and progression. Furthermore, there is a significant association between PDE4D7 expression and presence of the TMPRSS2-ERG gene fusion, the most common gene rearrangement in PCa which leads to overexpression of ERG and subsequent enhanced PCa aggressiveness.
Herein the inventors describe data obtained from prostate cancer cells in which expression of PDE4D7 has been specifically reduced using shRNA mediated knockdown. Surprisingly the inventors found that re-expressing PDE4D7 in these knockdown cells reduced proliferation. Furthermore expression of PDE4D7 was able to reduce resistance to drug treatments such as antiandrogen resistance, PARP inhibitor resistance or chemotherapy resistance. Based on these data the inventors concluded that increasing expression or increasing PDE4D7 phosphodiesterase activity reduces therapy resistance and may be used to treat therapy resistant prostate cancers.
Therefore in a first aspect the invention relates to a product for use in the treatment, prevention or amelioration of prostate cancer by reducing therapy resistance in a subject in need thereof, wherein the product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7. Alternatively the invention relates to a method of treating, preventing or ameliorating prostate cancer in a subject in need thereof, the method comprising administering to the subject product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7 and is selected from: a polynucleotide encoding a peptide having a sequence according to SEQ ID NO: 2 or a variant thereof, wherein the variant is a polynucleotide encoding a peptide having a sequence with at least 90% sequence homology with SEQ ID NO: 2 and wherein the peptide has phosphodiesterase activity; or a polynucleotide comprising a nucleotide sequence as defined in SEQ ID NO: 1 or a variant thereof, wherein the variant is a polynucleotide comprising a nucleotide sequence with at least 90% sequence homology to SEQ ID NO: 1 and wherein the polynucleotide encodes a peptide that has phosphodiesterase activity; or a peptide comprising a peptide sequence as defined in SEQ ID NO: 2 or a variant thereof, wherein the variant is a peptide comprising a peptide sequence with at least 90% sequence homology with SEQ ID NO: 2 and wherein the peptide has phosphodiesterase activity; or a peptide comprising a peptide sequence encoded by a polynucleotide comprising a nucleotide sequence as defined in SEQ ID NO: 1, or a variant thereof, wherein the variant is peptide comprising a peptide sequence encoded by a polynucleotide comprising a nucleotide sequence with at least 90% sequence homology to SEQ ID NO: 1 and wherein the polynucleotide encodes a peptide that has phosphodiesterase activity; or a compound as defined in formula I
Figure imgf000016_0001
Formula I wherein
Ri is H, (Cl-4)alkyl or (Cl-4)alkyloxy;
R2 and Re are independently selected from H and;
Rs, R4 and R5 are independently selected from H, halogen, CN, (Ci-4)alkyl and (Ci-4)alkyloxy, the (Ci- 4)alkyl and (Ci-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
R7, Rs, and Rio are independently selected from H and F;
Rs is selected from (Cl-4)alkyl, (Cl-4)alkyloxy, CN and halogen, the (Cl-4)alkyl and (Cl-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros; or a peptide having a sequence comprising or consisting a sequence selected from SEQ ID Nos: 24, 25, 26 or 27, or a polynucleotide encoding a peptide having a sequence comprising or consisting a sequence selected from SEQ ID Nos: 24, 25, 26 or 27.
As has been set out above, the present invention concerns in one aspect phosphodiesterase 4D7 (PDE4D7) for use as a therapeutic strategy. The term “phosphodiesterase 4D7” or “PDE4D7” relates to the splice variant 7 of the human phosphodiesterase PDE4D, i.e. the human phosphodiesterase PDE4D7 gene, preferably to the sequence as defined in Genbank Accession No: AF536976 (version AF536976.1, GE22901883 as of 3 Mar. 2009), more preferably to the nucleotide sequence as set forth in SEQ ID NO: 1, which corresponds to the sequence of the above indicated Genbank Accession number of the PDE4D7 transcript and corresponding protein accession number AAN10118 (version AAN10118.1), and also relates to the corresponding amino acid sequence as set forth in SEQ ID NO: 2, which corresponds to the sequence of the above indicated Genbank Accession number of the PDE4D7 polypeptide encoded by the PDE4D7 transcript. The term also comprises nucleotide sequences showing a high degree of homology to PDE4D7, e.g. nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 1, or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 2, or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 2, or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO: 1.
The term “human phosphodiesterase PDE4D7 gene”, “PDE4D7 gene” or “PDE4D7 marker gene” as used herein relates to the gene encoding phosphodiesterase 4D. Preferably, the term relates to a gene expressing phosphodiesterase 4D as splice variant 7, e.g. the specific exon combination as defined in Genbank Accession No: AF536976 (version AF536976.1, GE22901883 as of 3 Mar. 2009) or as set forth in SEQ ID NO: 1. The term also relates to DNA molecules derived from mRNA transcripts encoding phosphodiesterase 4D spliced as variant 7, preferably cDNA molecules.
The term “phosphodiesterase 4D7”, “phosphodiesterase 4D7 protein”, or “PDE4D7 protein” as used herein relates to the 4D7 splice variant encoded protein as defined herein above. Preferably, the term relates to a protein encoded by the phosphodiesterase 4D gene as splice variant 7, e.g. the specific exon combination as defined in Genbank Accession No: AF536976 (version AF536976.1, GE22901883 as of 3 Mar. 2009) or as set forth in SEQ ID NO: 2. The term also relates to proteins or polypeptides derived from the protein encoded by the PDE4D7 gene and which have been modified by addition of additional amino acid(s) or other modifications such as sugar residues, tags, phosphorylation, acetylation, methylation, ubiquitination or other modifications, or has been modified by the removal of one or more amino acids such as but not limited to a signal peptide.
The present invention is prompted by the inventors finding that re-introducing PDE4D7 in PDE4D7 knockdown prostate cancer cells has several beneficial effects such as reduction of cancer cell proliferation and reversal of therapy resistance. It is envisaged that such effect can thus be established in two manners: 1) by increasing the expression of PDE4D7 protein, that is, by increasing the amount of phosphodiesterase 4D7 protein in the prostate cancer cells or indirectly by increasing the expression level of the PDE4D7 isoform of the PDE4D gene, that is, by promoting transcription of the PDE4D gene isoform 7 or by otherwise increasing the amount of PDE4D7 mRNA; or 2) by increasing the activity of PDE4D7 enzyme, for example by removing or inhibiting antagonists of the PDE4D7 protein enzymatic activity, or stimulating agonists of the PDE4D7 protein enzyme activity, or by introducing additional protein (directly or indirectly), by introducing an active form of PDE4D7 protein or by a compound directly or indirectly engaging with PDE4D7 to active it.
Therefore, the present disclosure broadly defines a product for inducing the expression of PDE4D7 or promoting the activity of phosphodiesterase 4D7. In an aspect the product is a compound or composition. When used herein the term compound may refer to a chemical compound (also referred to as “small molecule therapy”), a biological such but not limited to a polynucleotide, a polypeptide, or a sugar polymer.
When used herein the term composition may refer to a mixture of compounds or a combination of a compound with adjuvants. A composition may be a solution or a dry composition.
The inventors envision several ways to achieve increased PDE4D7 expression or phosphodiesterase 4D7 activity in a subject, some non-limiting examples are listed below, however it is understood that the disclosure extends to any method or compound known to the skilled person to achieve increased expression of PDE4D7 or increased activity of the phosphodiesterase 4D7 isoform. Thus in an aspect of the disclosure the product is selected from: a gene therapy for inducing PDE4D7 expression; a compound that induces PDE4D7 expression; a compound directly stimulating or promoting phosphodiesterase 4D7 activity; a compound indirectly stimulating or promoting phosphodiesterase 4D7 activity; an inhibitor of a transcriptional inhibitor of PDE4D7; an inhibitor of an inhibitor of phosphodiesterase 4D7 enzymatic activity mRNA encoding a phosphodiesterase 4D7 protein; a phosphodiesterase 4D7 protein; a phosphodiesterase 4D7 activity promoting amyloid beta peptide.
As part of the invention the following products are envisioned to increase expression or activity of PDE4D7:
1) Directly introducing a protein with PDE4D7 activity. Therefore in an embodiment the invention describes a polynucleotide encoding a peptide having a sequence according to SEQ ID NO: 2 or a variant thereof, wherein the variant is a polynucleotide encoding a peptide having a sequence with at least 90% sequence homology with SEQ ID NO: 2 and wherein the peptide has phosphodiesterase activity. SEQ ID NO: 2 describes the amino acid sequence of the PDE4D isoform 7. Therefore a peptide comprising or consisting of a amino acid sequence as defined in SE QID NO: 2 (or a variant thereof as defined herein) can directly be introduced in a cell. Methods for introducing a peptide in a cell are known to the skilled person and include but are not limited to using cell penetrating peptides and nanoparticles.
2) Introducing a polynucleotide encoding a protein with PDE4D7 activity. Therefore in an embodiment the invention describes a polynucleotide comprising a nucleotide sequence as defined in SEQ ID NO: 1 or a variant thereof, wherein the variant is a polynucleotide comprising a nucleotide sequence with at least 90% sequence homology to SEQ ID NO: 1 and wherein the polynucleotide encodes a peptide that has phosphodiesterase activity. 3) a compound as defined in formula I
Figure imgf000019_0001
Formula I wherein
Ri is H, (Cl-4)alkyl or (Cl-4)alkyloxy;
R2 and Re are independently selected from H and;
Rs, R4 and R5 are independently selected from H, halogen, CN, (Ci-4)alkyl and (Ci-4)alkyloxy, the (Ci- 4)alkyl and (Ci-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
R7, Rs, and Rio are independently selected from H and F;
Rs is selected from (Cl-4)alkyl, (Cl-4)alkyloxy, CN and halogen, the (Cl-4)alkyl and (Cl-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros.
4) a peptide having a sequence comprising or consisting a sequence selected from SEQ ID Nos: 24, 25, 26 or 27, or a polynucleotide encoding a peptide having a sequence comprising or consisting a sequence selected from SEQ ID Nos: 24, 25, 26 or 27.
When used herein, a gene therapy for inducing PDE4D7 expression refers to a method to introduce a nucleotide encoding the phosphodiesterase 4D7 isoform in a cell, preferably a cell of a subject having prostate cancer, more preferably in a prostate cancer cell or a metastasis thereof. The nucleotide may for example be a viral vector or a non-viral vector, however it is understood that any nucleotide which can be introduced in a cell of a patient and express PDE4D7 may be used. For example the nucleotide may also be an mRNA encoding PDE4D7 or a variant of PDE4D7. Nucleotides such as mRNA may be expressed in a tumor cell in vivo using lipid nanoparticles (LNP). Suitable LNP formulations are known to the skilled person.
Viral vectors are a known strategy for gene therapy, typically viral-vector gene therapies use modified viruses as drug-delivery vehicles to introduce specific DNA or RNA sequences into cells. The vector is packaged using the viral proteins allowing infection of cells and expression of its viral genome. Typically the viral vector is modified such that no new viral particles can be produced upon infection of the target cell. Non limiting examples include retroviruses (RV), adenoviruses (AV), adeno- associated viruses (AAV), lentiviruses (LV), and herpes simplex viruses (HSV). Other ways to use viral particles or viral vectors to introduce the PDE4D7 encoding nucleotide are known to the person skilled in the art and also envisaged to be encompassed by the invention.
Alternatively to viral vector based gene therapy, a non-viral vector may be used. These vectors typically contain a promotor to drive expression of the relevant construct (e.g. PDE4D7), and typically require some means to introduce the vector into the target cell. Means to deliver a non-viral vector to a target cell in a subject are known to the skilled person, and for example reviewed in Ramamoorth M, Narvekar A. Non viral vectors in gene therapy- an overview. J Clin Diagn Res. 2015 Jan;9(l):GE01-6 (hereby incorporated by reference in its entirety). For example, nanoparticles can be used to encapsulate the vector. Non limiting examples are lipid based nanoparticles, peptide based nanoparticles, cationic lipid based nanoparticles, apolipoprotein based nanoparticles, (synthetic) polymer based nanoparticles such as polyethylenimine (PEI), chitosan, polylactate, polylactide, polyglucoside, denriomer or polymethracylate based nanoparticles. When used herein a nanoparticle is a small particle which can be used a sa carrier to deliver a cargo (payload) in a patient. Preferably the cargo is the product as broadly defined herein. Therefore a nanoparticle can be used to deliver the product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7 to a site in patient, e.g. a cell, tissue or organ. Other ways to use non- viral vectors to introduce the PDE4D7 encoding nucleotide are known to the person skilled in the art and also envisaged to be encompassed by the invention.
Therefore in an embodiment the gene therapy is selected from: a viral vector capable of expressing PDE4D7 in a subject or a non-viral vector capable of expressing PDE4D7 in a subject. In an embodiment the product is delivered using a nanoparticle.
When used herein the term vector is used to indicate any particle (e.g., a plasmid, a cosmid, a Lambda phage) used as a vehicle to artificially carry a foreign nucleic sequence - usually DNA - into another cell, where it can be replicated and/or expressed.
When used herein a compound that induces PDE4D7 expression is intended to refer to any biological or chemical compound that is able to increase the expression of PDE4D7. This may for example be achieved by promoting transcription of the PDE4D7 gene or inhibiting degradation of the phosphodiesterase 4D7 isoform protein. For example the compound may engage a signal transduction pathway to indirectly promote transcription of the PDE4D7 or directly interact with the promoter region of the genomic DNA to promote transcription of the PDE4D7 isoform.
When used herein promoting transcription of PDE4D7 refers to increasing the transcription of PDE4D7 resulting in a higher amount of PDE4D7 mRNA transcripts, and/or preferably in a higher amount of phosphodiesterase 4D7 isoform protein in the cell. The promoting may be specific for PDE4D7, specific for all PDE4D isoforms, specific for all PDE4 or even all PDE family members. In other words, increasing expression of PDE4D7 does not exclude that other PDE4D isoforms, other PDE4 family members or even other PDE family members are also increased in expression. When used herein, a compound directly stimulating or promoting phosphodiesterase 4D7 activity is intended to refer to a compound which directly engages with the phosphodiesterase 4D7 isoform protein enzymatic activity. Without wishing to be bound be theory, it is theorized that PDE proteins such as the PDE4D7 protein can exist in an active and inactive conformation, wherein activity can be induced by other compounds through interaction with the PDE protein, for example by promoting an active conformation of the protein or by blocking or preventing binding of inhibitors, or by modifying the protein to promote activity (for example by phosphorylation or other known protein modifications). When used herein, enzymatic activity when referring to a phosphodiesterase refers to catalysis of the hydrolysis of cAMP and/or cGMP.
When used herein, a compound indirectly stimulating or promoting phosphodiesterase 4D7 activity is intended to refer to a compound which indirectly engages with the phosphodiesterase 4D7 isoform protein enzymatic activity. Similarly as above it is envisaged that compounds may induce activators of PDE4D7 or block inhibitors form PDE4D7 activity and thus indirectly affect the protein’s enzymatic activity. The person skilled in the art is aware of methods to determine PDE or specifically PDE4D7 activity. For example, commercial products are available to assay for PDE activity, and methods have for example been described in Blair et al. Measuring cAMP Specific Phosphodiesterase Activity: A Two-step Radioassay. Bio Protoc. 2020 Apr 5;10(7):e3581, hereby incorporated in its entirety by reference.
When used herein, an inhibitor of a transcriptional inhibitor of PDE4D7 refers to a compound capable of blocking an inhibitor of PDE4D7 gene transcription. The inbitior of PDE4D7 gene transcription may be a direct inhibitor capable of binding the genomic DNA and preventing or reducing gene transcription or an indirect inhibitor which through downstream actions reduces or inhibits transcription of the PDE4D7 gene.
When used herein an inhibitor of an inhibitor of phosphodiesterase 4D7 enzymatic activity refers to a compound that can block an inhibitor of phosphodiesterase isoform 4D7 protein activity. For example the compound may function by degrading the inhibitor, preventing the inhibitor from engaging with PDE4D7 or by inhibiting the activity of the inhibitor.
When used herein, mRNA encoding a phosphodiesterase 4D7 protein refers to a RNA molecule from which the protein corresponding to SEQ ID NO: 2 or a substantially similar protein can be translated. The mRNA molecule generally comprises non translated elements such as 5’ and 3’ UTRs. It is anticipated that by direct introduction of PDE4D7 mRNA into the target cell (e.g. a prostate cancer cell in a subject) PDE4D7 can by upregulated. Method of delivering mRNA to a target cell are known to the skilled person, for example using nanobodies as described above.
When used herein a phosphodiesterase 4D7 protein refers to a compound comprising PDE4D7 protein or a biosimilar, or a constitutively active variant thereof, having phosphodiesterase activity. It is anticipated that by direct introduction of PDE4D7 protein into the target cell (e.g. a prostate cancer cell in a subject) PDE4D7 activity can be increased. Method of delivering protein to a target cell are known to the skilled person, for example using nanobodies as described above.
When used herein, a phosphodiesterase 4D7 activity promoting peptide refers to a peptide that is able to increase activity of the PDE4D7 enzyme. Non limiting examples are Amyloid beta (Abeta) peptides, which have been demonstrated to specifically increase long PDE4D isoforms such as PDE4D7. When used herein Amyloid beta, also known as Ap or Abeta, refers to peptides of 37-49 amino acids (Chen et al. Acta Pharmacol Sin 38, 1205-1235 (2017)) that are the main component of the amyloid plaques found in the brains of people with Alzheimer's disease. Amyloid beta peptide (A ) is produced through the proteolytic processing of a transmembrane protein, amyloid precursor protein (APP), by P- and y-secretases.
Therefore the phosphodiesterase 4D7 activity promoting peptide is preferably a Abeta peptide or derivative thereof. When used herein a Abeta peptide or derivative thereof is characterized as a peptide having a length of 16 to 50 amino acids and comprising an amino acid sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence defined by SEQ ID NO: 24 below (Abeta core peptide 1), more preferably comprising an amino acid sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence defined by SEQ ID NO: 25 below (Abeta core peptide 2). Non limiting examples are provided by Abeta 42 (SEQ ID NO: 26) and Abeta 40 (SEQ ID NO: 27). Therefore in an embodiment the Abeta peptide or derivative thereof is characterized as a peptide having a length of 42 to 50 amino acids and comprising an amino acid sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence defined by SEQ ID NO: 26, or a peptide having a length of 40 to 50 amino acids and comprising an amino acid sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence defined by SEQ ID NO: 27.
SEQ ID NO: 24 Abeta core peptide 2
HDSGYEVHHQKLVFFAEDVGSNKGAIIG SEQ ID NO: 25 Abeta core peptide 2 FRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMV SEQ ID NO: 26 Abeta 42 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA SEQ ID NO: 27 Abeta 40 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV The inventors describe herein several advantages for a treatment based on increasing PDE4D7 expression or activating phosphodiesterase 4D7 in a cell, such as reducing cancer cell proliferation. Moreover the inventors provide experimental data strongly suggesting that increasing PDE4D7 expression or activating phosphodiesterase 4D7 in a cell may help to overcome therapy resistance, or at least makes therapy resistant tumor cells more responsive to therapeutics, such as, among others, antiandrogens, PARP inhibitors and taxanes. Therefore in an embodiment the treatment, prevention or amelioration comprises reducing a therapy resistance.
When used herein reducing therapy resistance refers reducing or avoiding resistance to said therapy which has developed or may develop in a cancer cell. As such, co-adminstering the compound (product) that induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7 together with a therapy will unexpectedly increase the effectiveness of the therapy, in addition to the beneficial effects observed from the compounds derscibed herein.
Therefore in an embodiment the therapy resistance is antiandrogen resistance.
Antiandrogens may be androgen receptor antagonists. Non limiting examples of steroidal antiandrogens comprise 17a-Hydroxy progesterone derivatives such as, Chlormadinone acetate, Cyproterone acetate, Megestrol acetate, Osaterone acetate; 19-Norprogesterone derivatives such as Nomegestrol acetate; 19- Nortestosterone derivatives such as Dienogest, Oxendolone; 17a-Spirolactone derivatives such as Drospirenone, Spironolactone, Others such as Medrogestone. Non-limiting examples of nonsteroidal antiandrogens comprise Bicalutamide, Flutamide, Nilutamide, Apalutamide, Darolutamide, Enzalutamide, Proxalutamide, Cimetidine and Topilutamide.
Antiandrogens may be androgen synthesis inhibitors. Non-limiting examples of CYP17A1 inhibitors such as Abiraterone acetate, Ketoconazole, Seviteronel; CYP11A1 (P450scc) inhibitors such as Aminoglutethimide; 5a-Reductase inhibitors such as Alfatradiol, Dutasteride, Epristeride, Finasteride, Saw palmetto extract.
Antiandrogens may be antigonadotropins. Non-limiting exmaples are estrogens such as estradiol (and its esters), ethinylestradiol, conjugated estrogens, diethylstilbestrol; GnRH analogues such as GnRH agonists like goserelin, leuprorelin, GnRH antagonists like cetrorelix; Progestogens such as chlormadinone acetate, cyproterone acetate, gestonorone caproate, medroxyprogesterone acetate, megestrol acetate.
Therefore in an embodiment the therapy resistance is resistance to an androgen receptor antagonist, an androgen synthesis inhibitor or an antigonadotropin, preferably an androgen receptor antagonist, an androgen synthesis inhibitor or an antigonadotropin as listed above.
In a further preferred embodiment the therapy resistance is resistance to Enzalutamide, Flutamide, Nilutamide, Bicalutamide, Topilutamide, Apalutamide, Darolutamide, Cimetidine, ketoconazole, Proxalutamide (GT-0918), Seviteronel (VT-464), or Abiraterone. In a further preferred embodiment the therapy resistance is resistance to Enzalutamide.
In an embodiment the therapy resistance is PARP inhibitor resistance.
The PARP inhibitor resistance may be resistance to Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, Pamiparib (BGB-290), Rucaparib, CEP 9722, E7016, Iniparib or 3 -Aminobenzamide. Therefore in an embodiment the therapy resistance is resistance to a PARP inhibitor as listed above. In an embodiment the therapy resistance is resistance to a PARP inhibitor selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, Pamiparib (BGB-290), Rucaparib, CEP 9722, or E7016. In an embodiment the therapy resistance is resistance to Olaparib.
In an embodiment the therapy resistance is resistance to a chemotherapeutic agent. A chemotherapeutic agent may be an alkylating agent. Non-limiting examples of alkylating agents are Cyclophosphamide, Mechlorethamine, Chlorambucil, Melphalan, Dacarbazine, Nitrosoureas, and Temozolomide. Achemotherapeutic agent may be an anthracycline. Non-limiting examples of anthracy clines are Daunorubicin, Doxorubicin, Epirubicin., Idarubicin, Mitoxantrone, and Valrubicin. A chemotherapeutic agent may be a taxane. Non-limiting examples of taxanes are Paclitaxel, Docetaxel, Abraxane, and Taxotere. A chemotherapeutic agent may be ahistone deacetylase inhibitor. Non limiting examples of histone deacetylase inhibitors are Vorinostat and Romidepsin.A chemotherapeutic agent may be a topoisomerase inhibitor. Non-limiting examples of topoisomerase inhibitors are Irinotecan, Topotecan, Etoposide, Teniposide, and Tafluposide. A chemotherapeutic agent may be a kinase inhibitor. Non-limiting examples of kinase inhibitors are Bortezomib, Erlotinib, Gefitinib, Imatinib, Vemurafenib, and Vismodegib. A chemotherapeutic agent may be a nucleotide analog or precursor analog. Non-limiting examples of nucleotide analogs or precursor analogs are Azacitidine, Azathioprine, Capecitabine, Cytarabine, Doxifluridine, Fluorouracil, Gemcitabine, Hydroxyurea, Mercaptopurine, Methotrexate, and Tioguanine. A chemotherapeutic agent may be a platinum based agent. Non-limiting examples of platinum based agents are Carboplatin, Cisplatin, and Oxaliplatin. A chemotherapeutic agent may be a retinoid. Non-limiting examples of retinoids are Tretinoin, Alitretinoin, and Bexarotene. A chemotherapeutic agent may be a vinca alkaloid or derivative. Non-limiting examples of vinca alkaloids or derivatives are Vinblastine, Vincristine, Vindesine, and Vinorelbine.
Therefore in an embodiment the therapy resistance is resistance to an a chemotherapeutic agent as listed above. In an embodiment the resistance to an a chemotherapeutic agent is resistance to Paclitaxel, Docetaxel, Abraxane, Taxotere, Cabazitaxel, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Estramustine phosphate, Alestramustine, Atrimustine, Cytestrol acetate, Estradiol mustard, Estramustine, Estromustine, ICI-85966, or Phenestrol. In an embodiment the resistance to an a chemotherapeutic agent is resistance to Ocetaxel, Cabazitaxel, Mitoxantrone, Estramustine.
The present invention further provides that an additional therapy is administered to the subject, preferably a therapy to which resistance is avoided or reversed by the product as defined herein.
Therefore in an embodiment the treatment, prevention or amelioration further comprises administering an antiandrogen. The antiandrogen may be selected from the antiandrogens described above. In an embodiment the treatment, prevention or amelioration further comprises administering an antiandrogen selected from Enzalutamide, Flutamide, Nilutamide, Bicalutamide, Topilutamide, Apalutamide, Darolutamide, Cimetidine, ketoconazole, Proxalutamide (GT-0918), or Seviteronel (VT- 464), or Abiraterone. In an embodiment the treatment, prevention or amelioration further comprises administering Enzalutamide.
Therefore the treatment, prevention or amelioration further comprises administering a PARP inhibitor. The PARP inhibitor may be selected from the PARP inhibitors described above. In an embodiment the treatment, prevention or amelioration further comprises administering a PARP inhibitor selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, Pamiparib (BGB-290), Rucaparib, CEP 9722, or E7016. In an embodiment the treatment, prevention or amelioration further comprises administering Olaparib.
Therefore in an embodiment the treatment, prevention or amelioration further comprises administering a chemotherapeutic agent. The chemotherapeutic agent may be a chemotherapeutic agent as listed above. In an embodiment the treatment, prevention or amelioration further comprises administering a chemotherapeutic agent selected from: Paclitaxel, Docetaxel, Abraxane, Taxotere, Cabazitaxel, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Estramustine phosphate, Alestramustine, Atrimustine, Cytestrol acetate, Estradiol mustard, Estramustine, Estromustine, ICI- 85966, or Phenestrol. In an embodiment the treatment, prevention or amelioration further comprises administering a chemotherapeutic agent selected from to Ocetaxel, Cabazitaxel, Mitoxantrone, Estramustine.
In an embodiment the product as broadly described herein is administered to a subject in case the subject demonstrates therapy resistance. For example the therapy resistance is androgen deprivation resistance or PARP inhibitor resistance. In such cases it is very likely that PDE4D7 expression is reduced and that the subject may benefit form the product as broadly described herein. In an embodiment the product as broadly described herein is adminetered to a subject in case the tumor from the subject has low PDE4D7 expression, as it is envidsioned that in such cases it is very likely that PDE4D7 expression is reduced and that the subject may benefit form the product as broadly described herein.
It is further envisioned that prior to administering a product as broadly described herein a tumor or tissue sample of the patient is provided and analyzed for PDE4D7 expression. Thus, in an embodiment, the invention relates to a product for use in the treatment, prevention or amelioration of prostate cancer in a subject in need thereof, wherein the product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7, wherein the treatment, prevention or amelioration comprises providing a sample from the subject and determining the expression level of PDE4D7. Alternatively the invention relates to a method of treating, preventing or ameliorating prostate cancer in a subject in need thereof, the method comprising:
- providing a sample from the subject;
- determining the expression level of PDE4D7 in the sample; and
- administering to the subject the product a broadly defined herein. Instead of determining the expression levels of PDE4D7 the enzymatic activity of PDE4D7 protein may be determined. In an embodiment the product is administered to the subject when a low expression level of PDE4D7 is observed in the sample. In an embodiment an alternative treatment (e.g. not the product as defined herein) is administered to the subject when a high expression level of PDE4D7 is observed in the sample. The alternative treatment may for example be a conventional treatment option for prostate cancer. Preferably the alternative treatment in patients with localized disease is active surveillance (instead of active treatment). Preferably the alternative treatment in patients with non-localized disease is conventional / standard-of-care treatment or treatment de-intensification.
In an embodiment the sample is a tumor sample or a tissue sample from the subject, preferably the sample is a tumor sample, more preferably a prostate cancer tumor sample. For example the tumor sample may be a biopsy or culture obtained from the tumor.
The skilled person will be easily able to determine whether an expression level is deemed “high” or "low”, for example by creating a reference library of prostate tumor samples and determine PDE4D7 expression it can easily be determined what the overall range of PDE4D7 specific expression levels are found in tumor samples and which values correspond to a high value (e.g. above the median value) and which values correspond to a low value (e.g. below the median value).
In an alternative aspect of the invention is provided a compound for use in the treatment, prevention or amelioration of prostate cancer in a subject in need thereof, wherein the compound is an ATR inhibitor or an metabolic inhibitor. Preferably the compound is adminstered if:
- high expression of PDE4D7 is observed in a tumor;
- low expression of PDE4D7 is observed but PDE4D7 activity cannot be sufficiently increased to re-sensitize tumors to treatment with inhibitors; or
- in metastatic tumors.
The data presented here in examples 1-3 relating to Ceralasertib (ATR inhibitor; see Figures 4D, 4E and 17) and metformin (metabolic inhibitor; see Figures 6 A and 6B) demonstrate that these inhibitors reduce tumor growth independent of PDE4D7 expression levels. Tumors which already demonstrate high levels PDE4D7 may not respond to the product as broadly described above. The same applies to metastatic tumors for which it may be challenging to increase PDE4D7 activity (compared to a localized tumor). Further there may be tumors where increasing PDE4D7 activity is technically challenging for other reasons, e.g. the tumor does not respond well to the product as broadly defined herein. For these cases it may be beneficial to use as an alternative treatment an ATR inhibitor or an metabolic inhibitor.
In an embodiment the compound is an ATR inhibitor. In an embodiment the ATR inhibitor is selected from RP-3500, Ceralasertib (AZD6738), AZ20, Elimusertib (BAY-1895344), Elimusertib (BAY-1895344) hydrochloride, SKLB-197, ETP -46464, Berzosertib (VE-822), Dactolisib (BEZ235), VE-821, Torin 2, or CGK 733. In a preferred embodiment the ATR inhibitor is Ceralasertib. In an embodiment the compound is a metabolic inhibitor. Suitable metabolic inhibitors are known to the skilled person and for example described in Lemberg et al., J Clin Invest. 2022 Jan 4;132(l):el48550 and Luengo et al., Cell Chemical Biology 24, September 21, 2017, ppi 161-1180. In an embodiment the metabolic inhibitor is selected from Methotrexate, Pemetrexed, 6-Mercaptopurine, 6- Thioguanine, Capecitabine, 5 -Fluorouracil, Gemcitabine, Cytarabine, Leflunomide, CB-839, PEG-BCT- 100 (ADIPEG20), AEB-1102, L-Asparaginase, TVB-2640, AG-120 (ivosidenib), IDH305, BAY1436032, FT-2102, AG-221 (enasidenib), AG-881, AZD3965, CPI-613, or Metformin. In a preferred embodiment the metabolic inhibitor is metformin.
The invention further provides a kit of parts comprising a product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7 as broadly defined herein. Therefore, in an embodiment the kit comprises a compound or composition wherein the compound or composition induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7. In an embodiment the product is selected from: a gene therapy for inducing PDE4D7 expression; a compound that induces PDE4D7 expression; a compound directly stimulating or promoting phosphodiesterase 4D7 activity; a compound indirectly stimulating or promoting phosphodiesterase 4D7 activity; an inhibitor of a transcriptional inhibitor of PDE4D7; an inhibitor of an inhibitor of phosphodiesterase 4D7 enzymatic activity mRNA encoding a phosphodiesterase 4D7 protein; a phosphodiesterase 4D7 protein; a phosphodiesterase 4D7 activity promoting peptide.
In an embodiment the gene therapy is selected from: a viral vector capable of expressing PDE4D7 in a subject or a non-viral vector capable of expressing PDE4D7 in a subject. In an embodiment the product is comprised in a nanoparticle.
In an embodiment the kit of parts further comprises an antiandrogen and/or a PARP inhibitor and/or a chemotherapeutic agent. In an embodiment the antiandrogen is selected from Enzalutamide, Flutamide, Nihitamide, Bicalutamide, Topilutamide, Apalutamide, Darolutamide, Cimetidine, ketoconazole, Proxalutamide (GT-0918), Seviteronel (VT-464), or Abiraterone. In an embodiment the PARP inhibitor is selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, Pamiparib (BGB-290), Rucaparib, CEP 9722, or E7016. In an embodiment the chemotherapeutic agent is selected from Paclitaxel, Docetaxel, Abraxane, Taxotere, Cabazitaxel, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Estramustine phosphate, Alestramustine, Atrimustine, Cytestrol acetate, Estradiol mustard, Estramustine, Estromustine, ICI-85966, or Phenestrol.
In a further aspect the invention relates to the ex vivo or in vitro use of a product that induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7as broadly defined herein to increase expression of PDE4D7 and/or activity of phosphodiesterase 4D7 in a cell. It is contemplated that any method, use or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and/or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.
Examples
Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention.
Example 1
Methods
Generation of stable shRNA transduced LNCaP cell lines
Stable shRNA-mediated lentiviral transduced LNCaP cell lines were generated by AMSBIO (Table 1). Briefly, shRNA-PDE4D7 was cloned into a lentivector and delivered into LNCaP clone FGC cells (ATCC) via lentivirus (MOI=10). Cells were maintained in puromycin-supplemented medium prior to selection of stably transduced cells. A stable shRNA-PDE4D7 LNCaP cell line was then re-transduced with lentivirus delivering PDE4D7 to generate a re-introduced cell line. Scrambled-shRNA was used as a control. All cells were cultured in RPMI 1640 medium (GibcoD), supplemented with 10% (v/v) foetal bovine serum, 1% (v/v) L-Glutamine and 1% (v/v) penicillin/streptomycin, and stored within a 37°C incubator with 5% CO2.
Table 1: LNCaP FGC cells transduced with lentiviral shRNA for different levels of PDE4D7 expression.
Figure imgf000028_0001
Plasmid DNA transfection
LNCaP WT and stable transduced cell lines were transiently transfected with pcDNA3.1-PDE4D7-VSV plasmid DNA construct using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific) according to manufacturer’s protocol. Cells were incubated for 24h post-transfection prior to further experiments. xCELLigence Real-Time Cell Analysis (RTCA)
Real-time cell growth was analysed using the xCELLigence RTCA system (Agilent), which measures real-time cell adhesion across gold microelectrode biosensors on 96 well electronic plates (E-plates) within a 37°C incubator with 5% CO2. Cellular adhesion influences electrical impedance which is converted into the cell index (CI) via RTCA software. Initial background measurements were recorded with 100 pL RPMI 1640 medium, followed by addition of 100 pL LNCaP cell suspension (10,000 cells/wells) for measurement of CI over time. Approximately 24h after seeding, cells were treated with specified concentrations of drugs. Negative controls containing either 0.1% DMSO or untreated cells were also included. CI was transformed to normalised CI via the RTCA software to timepoint of treatment.
Western Blotting
Cells were lysed in 3T3 lysis buffer (50 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 25 mM HEPES, 2.5 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100; pH 7.5) supplemented with cOmplete™ EDTA-free protease inhibitor cocktail (Sigma- Aldrich). Lysed cells were rotated end-over- end for 30 minutes at 4°C, then centrifuged for 10 minutes at 13,000 RPM at 4oC. Supernatants were collected and protein concentration determined via Bradford assay. Equal concentrations of protein samples were separated via SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes for western blot. Membranes were blocked in 5% (w/v) milk powder in TBS-T for Ih prior to primary and secondary antibody incubations. Primary antibodies used: anti-PDE4D5 1:5000 (Baillie lab), anti-PDE4D7 1:500 (Baillie lab), anti-PDE4D9 1:5000 (Baillie lab), anti-Pan- PDE4D 1:5000 (Baillie lab), anti-VSV 1:5000 (Abeam, #abl874), anti-GAPDH 1:5000 (Abeam, #ab8245), anti-AR 1:1000 (Abeam, #), anti-PSMA 1:1000, anti-E-cadherin 1:500 (Cell Signalling, #3195S), anti-TMPRSS2 1:1000 (Abeam #abl09131), anti-NKX3.1 1:1000 (Cell Signalling, #92998), anti-PSA 1:1000 (Abeam, #ab76113), anti-AR-V7 (Cell Signalling, #68492), anti-GR (Cell Signalling, #12041T). Secondary antibodies used: IRDye® 800CW Donkey-anti-Rabbit IgG 1:5000 (LI-COR, #926- 32213), IRDye® 680RD Donkey -anti-Goat IgG 1:5000 (LI-COR, #926-68074), IRDye 800CW Goat- anti-Human IgG 1:5000 (LI-COR, #926-32232), IRDye® 680RD Donkey -anti-Mouse IgG 1:5000 (LI- COR, #925-68072).
RNA analysis qRT-PCR
RNA was extracted using the RNeasy Mini Kit (QIAGEN) and one-step qRT-PCR was performed with the Superscript™ III Platinum™ One-Step qRT-PCR Kit (Thermo Fisher Scientific) as per manufacturers’ protocols. Experiments were carried out using the Step-One™ Real-Time PCR System (Thermo Fisher Scientific) at the following parameters: One cycle of 50oC for 30 minutes and 95oC for 5 minutes, followed by 45 cycles of 95oC for 15 seconds and 60oC for 45 seconds. Primers and probes were purchased from Integrated DNA Technologies (Table 2), with 400nM primer/200nM probe and Ing/ul RNA per reaction. Ct values were quantified using the 2-AACt method and statistical analysis was performed on AACt values.
Table 2: Primers and probe sequences used for RT-qPCR. PUM1, TBP, ACTB and HPRT1 were used as housekeeping genes. All probes were labelled 5' 6-FAM/ZEN/3' IBFQ.
Figure imgf000030_0001
NGS RNA sequencing
100 ng of total RNA was extracted from LNCaP cell lines and analysed via next generation RNA sequencing (RNAseq) using the NovaSeq 6000 S4 system (2x150 bp read length). RNAseq reads were mapped to the human reference genome (Ensembl 96) using STAR. Transcript per million (TPM) values were analysed between WT and shRNA transduced LNCaP cell lines and converted into z-scores. RNAseq data was further analysed via iPathwayGuide (Advaita Bio) to calculate the log2 fold change of genes associated with the prostate cancer pathway.
Immunofluorescence
LNCaP cells were seeded onto 13mm glass coverslips 24 hours prior to transfection as previously described. Cells were fixed with 4% (v/v) paraformaldehyde for 15 minutes, blocked and permeabilised with 10% donkey serum, 0.5% BSA and 0.2% Triton-X in PBS. Coverslips were incubated overnight with anti-PDE4D7 primary antibody (1:200, Baillie Lab), followed by incubation in Alexa-Fluor 488 Donkey -anti-Goat 1:500 secondary antibody (Thermo Fisher Scientific, #A-11055). Coverslips were mounted onto slides with Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich) and imaged via the ZEISS LSM 880 laser scanning microscope with 63x oil immersion objective.
Statistical Analysis
Values are presented as mean ± standard error of mean (S.E.M) for N>3, unless otherwise stated.
Statistical analyses were performed either via one-way ANOVA + Dunnett’s multiple comparison, two- way ANOVA + Sidak’s multiple comparison, or via unpaired t-test with assumed Gaussian distribution. P<0.05 was deemed significant (* = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001). All statistical analyses and models were performed via GraphPad Prism 9.
Results
Generation and sequencing of stable PDE4D7 -knockdown LNCaP
A stable, PDE4D7-knockdown LNCaP cell line was generated via shRNA-mediated lentiviral transduction. Several knockdown clones were analysed for PDE4D7 expression, and Pl cells revealed the most significant PDE4D7 mRNA-mediated downregulation via qPCR (Fig. 7) and was chosen alongside the WT and scrambled-shRNA LNCaP cell lines to look into the effects of PDE4D7 expression in prostate cancer. Proof of significant PDE4D7 knockdown at mRNA [Fig. 1 A] and protein level [Figs. IB] in the Pl LNCaP cell line compared to WT was obtained. The reduction in PDE4D7 protein expression was also confirmed using confocal microscopy [Fig. 1C] .
Analysis of alterations in genomic profiles between WT, scrambled and Pl cells were determined via NGS RNA seq. Top differentially expressed genes [Fig. ID] are shown as z-scores between WT and Pl cells, with many of these genes exhibiting significant downregulation in Pl LNCaPs. Advaita pathway analysis was performed on RNA seq data, and it identified that the top differentially expressed genes are implicated in PCa-related signalling pathways [Fig. IE], with logFC downregulated most significantly for KLK3, AR, SPINT1, TMPRSS2 and NKX3.1. To confirm that the RNAseq data was reflected at the protein level, western blotting for selected proteins was performed. Highly significant downregulation of all chosen proteins in the Pl cell line [Fig. IF] were observed in comparison to WT. Importantly, the scrambled-shRNA cell line showed no notable difference in expression of these proteins (Fig. 8).
PDE4D7-knockdown in LNCaP cells alters phenotype and confers resistance to enzalutamide Knockdown of PDE4D7 in LNCaP cell line Pl significantly enhanced growth characteristics of the cells. To confirm that the effect observed was due to PDE4D7-knockdown as opposed to integration of the lentivirus, xCELLigence growth assays were also performed on WT, Pl and the scrambled-shRNA knockdown (SC2) LNCaP cell lines. Real-time growth curves showed enhanced growth of Pl cells in comparison to both WT and SC2 [Fig. 2A], correlating with a significant difference in slope analysis. Importantly, no significant difference in growth characteristics were found between the WT and SC2 LNCaP cells.
Enzalutamide is an AR antagonist used in the treatment of advanced PCa (Menon and Higano, 2013). Given that significant down-regulation of PDE4D7 expression is a characteristic of androgen-insensitive prostate cancer (Henderson et al., 2014), we wanted to investigate the effects on the growth of WT and PDE4D7-silenced PCa cells following treatment with enzalutamide. To assess optimum treatment concentrations in WT and Pl LNCaP cells, a dose response experiment was performed using increasing drug concentrations and monitored via xCELLigence technology. All concentrations tested resulted in a significant decrease in WT LNCaP cell proliferation (0.1-30 pM), with IpM and above resulting in the least growth [Fig. 2B], In contrast, Pl LNCaP showed minimal reductions in cell proliferation upon increasing doses of enzalutamide [Fig. 2C], and even exhibited significantly enhanced growth in comparison to DMSO-control as concentration increased to 30pM. Overall, this data strongly suggests that PDE4D7-knockdown in LNCaP cells confers resistance to enzalutamide.
Given the significant difference of AR expression between WT and Pl cells [Fig. IE and F], we decided to test expression of the most relevant AR variants (AR-V7), however results did not reveal any obvious difference in expression [Fig. 2D], RNA seq analysis also identified CCND1 as a significantly downregulated gene in Pl cells compared to WT [Fig. IE],
Cyclin DI regulates androgen-dependent transcription and cell cycle progression [REF], Given that AR is downregulated in Pl cells, it is unsurprising therefore that CCND1 is also downregulated [Fig. 2F], Interestingly, however, cyclin Bl appears upregulated in Pl cells compared to WT [Fig. 2E], This could represent a compensatory mechanism in the cell, with upregulated expression of other cell cycle proteins to induce the evident enhanced proliferation.
PDE4D7 -knockdown in LNCaP cells confers resistance to P ARP -inhibition and is rescued upon reintroduction ofPDE4D7
Olaparib is an FDA-approved Poly (ADP -ribose) polymerase (PARP) inhibitor that is effective against LNCaP cells (Feiersinger et al., 2018). Ceralasertib is an ataxia-telangiectasia mutated and Rad3-related (ATR) inhibitor (AZD6738), that has been suggested to enhance synergistic effects in combination with olaparib in mCRPC (Lloyd et al., 2020). In light of this, we wanted to determine whether PDE4D7- knockdown affected LNCaP cells response to either of these treatments. Using real-time growth analysis, LNCaP WT proliferation decreased in a dose-dependent manner with olaparib [Fig. 3A], whereas the effect on PDE4D7-deficient LNCaPs was minimal [Fig. 3B], Evaluation of growth rate via slope analysis yielded similar data with significant reductions in WT growth at 3, 10 and 30pM olaparib, but with unexpected reduction in slope at low concentrations (0.3 and 1 pM) in cells where PDE4D7 was knocked down. It is notable, however, that cells with reduced PDE4D7 expression were resistant to olaparib at 10 and 30pM [Fig. 3B],
Cleavage of PARP is required for late-stage apoptosis, therefore analysis of PARP/cPARP ratio can be used to assess whether cells’ induction of apoptosis [REF], Here, there is a reduction in PARP/cPARP ratio between WT and Pl cells [Fig 3C].
Given that PDE4D7-knockdown LNCaPs exhibited enhanced growth compared to WT cells [Fig. 2A] and were resistant to PARP inhibition via olaparib [Fig. 3C], we wanted to determine whether we could rescue the phenotype through re-introduction of PDE4D7 into the Pl cell line. Initially, real-time growth was evaluated for Pl and P1-PDE4D7+/+ cells with significant downregulation in neoplastic growth observed upon reintroduction of PDE4D7 to Pl cells [Fig. 3D], Furthermore, P1-PDE4D7+/+ cells were more sensitive to olaparib treatment [Fig. 3E], as was previously apparent in the WT LNCaP cells, with a statistically significant downregulation in growth recorded when compared to both olaparib-treated Pl cells, and DMSO-treated P1-PDE4D7+/+ cells. This data re-confirms the role of PDE4D7 is blunting PCa cell growth and promoting susceptibility to clinically relevant PCa chemotherapeutics.
PDE4D7-knockdown in LNCaP cells does not affect response to ATR inhibition yet may limit docetaxel treatment response
Docetaxel is a DNA-damage inducing chemotherapy drug and we decided to utilise it to discover whether there is a difference between the ability of WT and Pl cells to repair DNA and hence survive upon treatment. Compared to DMSO, docetaxel significantly reduced growth of both WT and PDE4D7- knockdown LNCaPs at all concentrations (Figs. 4A and 4B, respectively], except for lOpM in Pl cells which appeared to have no effect. Given that lOpM was effective in WT but not Pl, we investigated how PDE4D7-reintroduction to Pl cells affected this. As with olaparib [Fig. 3D], P1-PDE4D7+/+ cells were more sensitive to docetaxel treatment than Pl [Fig. 4C], suggesting that a paucity of PDE4D7 promotes the ability to repair associated DNA damage and protects against induction of cell death.
Interestingly, whilst PDE4D7-knockdown conferred resistance of LNCaP cells to Olaparib at 10-30pM, these cells were extremely sensitive to ceralasertib at the same concentrations. Growth curves for WT and PDE4D7-knockdown LNCaP treated with increasing concentrations of ceralasertib showed similar trends with respect to sensitivity to the compound [Fig. 4D and 4E, respectively]. Analysis of curve slopes between 0-30h post-treatment yielded similar results for both cell types. In both cases, the most significant growth inhibition occurred at 10-30pM treatment. These results highlight that PDE4D7 silencing confers resistance to P ARP -inhibition via olaparib, however these cells are highly susceptible to ATR-inhibition via ceralasertib.
PDE4D7-knockdown in LNCaP cells affects response to many current anti-cancer compounds Many of the RNA seq genes identified as differentially expressed between WT and Pl cells are implicated in anti-androgen resistance. Signalling via the glucocorticoid receptor (GR) can bypass AR inhibition in CRPC patients leading to enhanced proliferation [REF], As opposed to most differential genes which appeared to be downregulated in Pl cells, GR expression was upregulated in Pl and confirmed via western blot as significant protein upregulation [Fig. 5A], We therefore decided to analyse the response to the GR antagonist mifepristone. Real-time growth analysis revealed that neither WT or Pl cell proliferation were reduced compared to DMSO from 0.3-10pM mifepristone treatment [Figs. 5B and 5C, respectively]. However, 30pM mifepristone successfully inhibited growth of WT LNCaPs yet at the same concentration had an opposing effect on Pl LNCaPs with significantly enhanced proliferation. Analysis of the growth curves show that for Pl LNCaPs with 30pM mifepristone [Fig. 5C: pink trace] initial growth was inhibited compared to lower concentrations, however this effect eventually wore off and the cells regained their proliferative potential.
Screening of the Prestwick library against DuCaP and DU 145 PCa cell lines revealed that anti-parasitic drugs show activity against these cells [Ralf data - to include?]. Repurposing of these drugs such as mebendazole has showed potential in advanced PCa treatment in combination with docetaxel [REF], Analysis of the effect of mebendazole on the WT and PDE4D7-knockdown LNCaPs showed significant differences between the cell lines responses. Interestingly, whilst WT cells showed significant downregulation of cell growth in a dose-dependent manner [Fig. 5D], the effect of Pl cells was far more substantial and more significant, with initiation of immediate cell death in the cells at both concentrations [Fig. 5E], This data suggests that PDE4D7-knockdown confers sensitivity to mebendazole in comparison to WT LNCaPs. cAMP dynamics in response to PDE4D7 knockdown and effect ofPDE4 activator
Genetically encoded cAMP -fluorescent resonance energy transfer (FRET) probes were used to investigate cAMP dynamics at subcellular regions in WT and Pl LNCaPs. CUTie cAMP FRET probes have been shown to have many advantages over conventional constructs [REF], CUTie probes targeting the plasmamembrane (AKAP-79) or cytosol (CYTO-pDUAL) were obtained from the Zaccolo lab (REF). Treatment with rolipram induced a downregulation of FRET ratio in the membrane compartment of WT LNCaPs, indicating an increase in cAMP concentration. The effect of a combination of forskolin (adenylate cyclase activator) and IBMX (non-specific PDE inhibitor) treatment which normally gives maximal response had no further effect on these cells, highlighting that PDE4 inhibition was responsible for maximising cAMP concentration at the membrane of WT LNCaPs [Fig. 6B], Pl LNCaPs showed no obvious difference in FRET ratio upon rolipram treatment, with a slight decrease in ratio/increase in intracellular cAMP upon forskolin/IBMX addition [Fig. 6D] . Given that PDE4D7 is known to be substantially membrane located [Fig. 1C] [REFs], inhibition via rolipram leads to upregulated cAMP at this locale in WT cells but not in Pl cells, as PDE4D7 has already been knocked down therefore basal cAMP levels are already likely to be elevated. Whilst PDE4D7 is also known to be in the cytosol, no observable difference was seen in WT and Pl LNCaPs upon rolipram treatment at this location, however forskolin/IBMX addition did appear to decrease FRET ratio/increase cAMP [Figs. 6A and 6C].
Metformin is a well-known drug used to treat type-2 diabetes, however its potential in treating numerous cancers has shown promising results [REF], With regards to PCa it remains controversial, where it has shown to enhance overall survival/time to recurrence, however no evidence of it treating/reducing the PCa tumours itself (He et al., 2019). Given that metformin is known to decrease cAMP production (Miller et al., 2013), we decided to test its effects between WT and Pl LNCaPs. Analysis of growth phenotype shows that metformin was successful at significantly reducing growth of WT cells at all concentrations [Fig. 6E], however significant inhibition was only observed in Pl cells at highest concentrations tested [Fig. 6F],
ML-R2 is a PDE4 longform activator compound that binds allosterically to the enzyme in order to mimic PKA phosphorylation in the UCR1 domain [REF], As PDE4D7 knockdown leads to an enhanced growth phenotype in LNCaPs, we decided to test whether this could be rescued using the PDE4 activator. Our results revealed that, whilst there was no effect on proliferation in WT LNCaPs at either concentration [Fig. 6G], ML-R2 addition caused inhibition of cell growth in Pl cells [Fig. 6H], Whilst PDE4D7-knockdown in LNCaPs leads to enhanced cell growth, activation of PDE4 in these cells has an inhibitory effect on their proliferative potential, highlighting again the importance of the PDE4 family, specifically PDE4D7, in driving PCa.
Example 2 Stable P1-PDE4D7+/+ LNCaP cell line were analyzed using cell index (N=3, Figure 9). Stable reintroduction resulted in expression of PDE4D7 protein as confirmed by Western blot. Stable reintroduction of PDE4D7 into Pl LNCaPs reduced real-time growth. Statistically significant downregulation of growth in P14D7+ LNCaPs was observed using cell index and slope analysis. It was confirmed that reintroduction of PDE4D7 in P1-PDE4D7 LNCaP cells reversed the knockdown phenotpy (N=3 completed just need to plot graph).
The PDE4 long form activator ML-R2 was introduced in WT, Pl and P14D7 cells and real-time growth of LNCaPs (N=3) was reviewed (Fig. 10). MLR2 has no effect on growth of WT LNCaPs, MLR2 attenuates growth of Pl LNCaPs and stable PDE4D7-reintroduction to Pl LNCaPs rescues phenotype of WT LNCaPs on MLR2 sensitivity.
Pl and P1+4D7 LNCaP cells were treated with Olaparib (N=3, Fig. 11). As a control, P1+4D7 cells were treated with DMSO. A+B) Reintroduction of PDE4D7 to Pl enhances sensitivity to Olaparib, however reintroduction of PDE4D7 alone also reduces growth to same extent.
Expression of PARP/cPARP in WT and Pl LNCaP +/- Olaparib/docetaxel treated cells was investigated (N=3, Fig. 12). Treatment with Olaparib/docetaxel increased PARP cleavage in WT LNCaPs correlating with induction of apoptosis (as seen in xCELLigence). No increase in cleaved PARP ratio in Pl LNCaPs correlating that these cells are not undergoing apoptosis in response to treatment (correlating with xCELLigence).
Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.
While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.
All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references. Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein) , readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.
Example 3
Methods
Patient cohorts and tissue samples
In total, 367 patients managed at two prostate cancer centers in Germany were included into this study. Of those, 363 patients had at least one adverse feature in pathology (PSM: positive surgical margins; SVI: seminal vesicle invasion; EPE: extra-prostatic extension; LNI: lymph node invasion; Gleason 4 component) and/or were classified into post-treatment intermediate or high risk of disease progression according to the clinical risk metrics CAPRA-S (24) and/or the EAU-BCR risk model (25) while 4 patients demonstrated a post-operatively rising PSA in the absence of any of the stated adverse features; all 367 patients experienced biochemical recurrence during post-treatment follow-up and were scheduled to salvage radiation therapy (SRT) of the pelvic bed with a total administered radiation dose between SO- 72 Gy over multiple fractions. Of the 367 patients 188 also received anti -androgen therapy at different time points (before, during or after SRT).
Two small biopsy punches (-1x2 mm) of a representative resected tumor area of 367 patients operated on between 1994-2011 were collected from the tumors index lesion. The local Institutional Review Boards approved the collection of patient tissue for clinical research.
Generation of stable PDE4D7 modified LNCaP prostate cancer cells
Stable shRNA mediated PDE4D7 knockdown LNCaP
LNCaP clone FGC (ATCC® CRL1740™) were cultured in RPMI 1640medium (Gibco™), supplemented with 10% (v/v) foetal bovine serum, 1% (v/v) L-Glutamine and 1% (v/v) penicillin/streptomycin, and stored within a 37°C incubator with 5% CO2. The transfer plasmid of the lentivirus transfection system was designed to stably express and shRNA RNA hairpin targeting the first coding exon of the PDE4D7 transcript. As a control, a scrambled nucleotide sequence of the PDE4D7 targeting shRNA was cloned into the transfer plasmid of the lentivirus. LNCaP clone FGC cells were infected with lentiviruses at MOI=10 in the presence of 10 pg/ml polybrene. Cells were maintained in puromycin-supplemented medium prior to selection and clonal expansion of stably transduced cells (for details see Supplementary Materials). RNA extraction and qPCR verification of gene knockdown were performed as described below.
Inducible PDE4D7 expression in PDE4D7 knockdown LNCaP Pl cells
PDE4D7 (GenBank ID AF536976) sequence was first subcloned into lentivirus vectors with CMV (LVR- 1001-pLV-CMV-PGK-Puro) and then into Tet-On inducible vector pLV-tet-on-3G-P2A-Puro, LNCaP-shRNA Pl cells were maintained in RPMI1640 medium supplemented with 10% (v/v) foetal bovine serum, 1% (v/v) L-Glutamine and 1% (v/v) penicillin/streptomycin, and infected with Tet-On inducible lentivirus at MOI=5-10 in the presence of 8 pg/ml polybrene. Cells were maintained in puromycin-supplemented medium prior to selection and clonal expansion of stably transduced cells (for details see Supplementary Materials). Cells transduced with Tet-On inducible lentivirus have been tested for the induction of PDE4D7 expression in the presence of 1 pg/ml Doxycycline. Expression of PDE4D7 has been estimated by RT-PCR.
Molecular Analysis
For downstream molecular analysis the following cell lines/clones were used: LNCaP FCG wildtype (WT); LNCaP transfected with scrambled shRNA control (clone SC2); LNCaP transfected with PDE4D7 targeting shRNA (clones Pl and w5.2), and LNCaP clone Pl transfected with Tet-On inducible PDE4D7 vector (clone P1-TET4D7).
RT-qPCR Analysis
- Patient sample biopsies:
To account for potential tumour heterogeneity the two tissue punches of the RP cohort were combined before nucleic acid extraction. A potential difference in tumour cellularity was addressed by normalisation of the PDE4D transcript expression to four reference genes. All used molecular laboratory methods including RNA extraction, quantitative real-time PCR (RT-qPCR), oligonucleotide primers/probes and statistical analysis were as previously described (21).
Cell lines: RNA was extracted from LNCaP cell lines using the RNeasy Mini Kit (QIAGEN) and one- step qRT-PCR was performed with the Superscript™ III Platinum™ One-Step qRT-PCR Kit (Thermo Fisher Scientific) as per manufacturers’ protocols. Experiments were carried out using the Step-One™ Real-Time PCR System (Thermo Fisher Scientific) at the following parameters: One cycle of 50°C for 30 minutes and 95°C for 5 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 45 seconds. Primers and probes were purchased from Integrated DNA Technologies (Supplementary Methods), with 400nM primer/200nM probe and Ing/pl RNA per reaction. Ct values were quantified using the 2'AACt method and statistical analysis was performed on AACt values. Calculation of Clinical and Genomic Risk Scores
Calculation of clinical risk score CAPRA-S
The post-surgical CAPRA-S risk score and its corresponding low (1), intermediate (2), high-risk (3) categories were calculated as described earlier (24). The EAU-BCR score and its two categories of low and high post-surgical risk of disease progression was determined as published (25).
Calculation of the PDE4D7 Score and PDE4D7 Score Categories
Generation of normalized PDE4D7 transcript expression was performed by subtracting the RT-qPCR Cq of the PDE4D7 transcript Cq from the averaged RT-qPCR Cq of the reference genes and transformed to the PDE4D7 score and its related PDE4D7 score classes (21). In multivariable analysis Cox and logistic regression analysis for various available biological and treatment related outcomes the PDE4D7 score was used as a continuous variable. For Kaplan-Meier survival analysis we transformed the normalized PDE4D7 transcript expression values to a percentile distribution (pPDE4D7 score) which allowed more efficient comparison with other genomic scores as all scores were distributed between 0-1 (see also below). Two cut-offs for the pPDE4D7 score were defined by AUROC (Area under the ROC curve) analysis with 5-year prostate cancer specific death after start of SRT as the dependent variable and the pPDE4D7 score as the independent variable. One cut-off (pPDE4D7>0.2) was defined as the point in the AUROC with maximum sensitivity and specificity. The cut-off was defined as max sensitivity (pPDE4D7>0.87). Consequently, the pPDE4D7 score classes stratified patients into three sub-cohorts (pPDE4D7 scores >0.87: ‘high’; pPDE4D7 scores >0.2 and <=0.87: ‘intermediate’, and pPDE4D7 scores <=0.2: ‘low’).
Calculation of the Cell Cycle Progression and the Genomic Prostate Score
To derive the risk scores for the two commercially available prognostic test which uses the CCP (Cell Cycle progression) 31 gene signature and GPS (Genomic Prostate Score) 12 gene signature we followed previously published protocols to infer the mxCCP and mxGPS scores from RNAseq expression data of the respective target and reference genes based on previously published formulas to calculate the risk scores (26). The derived scores were converted to percentile distributions (mxpCCP and mxpGPS, respectively). Where indicated we either used the mxCCP and mxGPS (AUROC analysis) or the mxpCCP and mxpGPS (Cox regression and Kaplan-Meier survival) for use in down-stream statistical analysis.
RNA sequencing was perfomred as described in Example 1.
Gene Set Enrichment Analysis (GSEA)
Gene Set Enrichment Analysis (GSEA) was performed as described here (https://www.gsea- msigdb.org/gsea/index.jsp). As input for the enrichment analysis the feature count matrices of the gene expression as derived from RNAseq were used.
Whole Genome Sequencing (WGS)
Approximately 100 ng gDNA per sample was randomly fragmented at 350 bp by ultrasonication.
Libraries were constructed following the TruSeq DNA Nano protocol (Illumina). In brief, sheared gDNA was end-prepped, dA-tailed, and enriched for fragments of approx. 350 bp through size selection with AMPure XP beads (Beckman Coulter). Indexing PCR for a total of 8 cycles was performed for the size- selected fragments, and the products were purified with AMPure XP beads. The quality of the final libraries was checked on the LabChip GX Nucleic Acid Analyzer (PerkinElmer). The concentration was determined by performing qPCR on the samples using the 2X KAPA HiFi HotStart ReadyMix (Kapa Biosystems) as mastermix and a dilution of PhiX Sequencing Control v3 as standard. High coverage sequencing was performed on the Illumina Novaseq 6000 system with the S4 flowcell and PE150 configuration.
Plasmid DNA transfection, eCELLigence Real-time Cell analysis, Western Blotting, and Immunofluorescence were performed as described in Example 1.
Data analysis and statistics
Multivariate Cox regression and Kaplan Meier analyses were applied to correlate the PDE4D7 score or the clinical metrics (CAPRA-S score, EAU-BCR risk score) and its categories to prostate cancer or allcause mortality in the total patient cohort (n=367). For statistical analysis the software package MedCalc (MedCalc Software v2.20 BVBA, Ostend, Belgium) was used using statistical tests as indicated in the main text of the manuscript and/or in the figure legends. For LNCaP data, values are presented as mean ± standard error of mean (S.E.M) or standard deviation (SD) for N>3, unless otherwise stated. Statistical analyses were performed either via one-way ANOVA + Dunnett’s multiple comparison, two-way ANOVA + Sidak’s multiple comparison, or via unpaired t-test with assumed Gaussian distribution. P<0.05 was deemed significant (* = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001). All statistical analyses and models were performed via GraphPad Prism 9.
Results
The PDE4D7 score is associated with survival outcomes in high-risk prostate cancer patients
All subjects enrolled in this study experienced post-surgical PSA relapse and were subsequently stratified for salvage radiation therapy (SRT). Pathological analysis of surgical specimens revealed pT3a or higher disease in nearly 80% of the 367 patients (Table 3). Extra-prostate extension and positive surgical resection margins were identified in 62.4% and 72.2% of cases, respectively. Based on the CAPRA-S risk score, the majority of patients (85%) were classified as intermediate risk (41.1%) or high risk (44.1%) for post-surgical disease progression. Over half of the patients (188/367) received androgen deprivation therapy (ADT) at some point during the study period. During a median follow-up of 103 months postprostatectomy, 11.7% of subjects died from prostate cancer and 17.4% died from any cause (Table 3). Multivariate Cox regression analysis was conducted to assess the association between the PDE4D7 score and prostate cancer-specific mortality (PCSM) after SRT initiation, adjusting for pathological prognostic variables (i.e., ISUP Gleason grade group, pathological pT stage, status of extra-prostate extension and surgical margins, seminal vesicle involvement, and lymph node invasion). As previously reported for biochemical recurrence as a clinical endpoint, we observed a strong inverse association between the PDE4D7 score and PCSM after SRT initiation (HR=0.37; 95% CI 0.23-0.58; logrank pO.0001) in the multivariable model that included clinical prognostic parameters (Table 2). To determine whether the PDE4D7 score provided independent prognostic information in the context of the genomic mxpCCP or mxpGPS signatures, these scores were separately included in the multivariable analysis. Interestingly, the mxpCCP score was not significantly associated with PCSM in this cohort (p=0.17), which may be due to limitations in inferring the risk score from RNAseq data (see methods). The mxpGPS signature demonstrated a borderline significant association with PCSM (HR=4.0; 95% CI 0.93-17.5; logrank p=0.06). In both analyses, the PDE4D7 score remained a significant variable in the multivariable model (p=0.003 and p=0.002). The PDE4D7 score was also tested in multivariable analysis for PCSM in combination with the clinical prognostic models CAPRA-S and EAU-BCR risk score. In both models, the PDE4D7 score remained the most significant predictor for PCSM after SRT (HR=0.36, p<0.0001;
HR=0.31, pO.OOOl, respectively).
The Kaplan-Meier (KM) analysis of pPDE4D7 percentile scores stratified patients into three sub-cohorts with significantly different survival rates (logrank p<0.0001; Figure 13A). Patients in the high pPDE4D7 score class had 100% survival rate after SRT, while those in the intermediate and low score classes had a survival rate of approximately 90% and below 50%, respectively, after 10 years post-SRT (Figure 1 A). All-cause mortality exhibited similar trends in this patient cohort (Figure 13B). The EAU-BCR Risk model stratified patients into low- and high-risk groups, with the low-risk group having a significantly better survival rate at 10 years post-radiation (HR=3.7; 95% CI 2.0-6.8; logrank p<0.0001; Figure 13C). The survival rate in the low-risk group was greater than 90%, while it was approximately 72% in the high-risk group. The CAPRA-S model did not show significant stratification between risk groups. To assess the predictive ability of the PDE4D7 score for 5-year PCSM after salvage radiation treatment, we conducted receiver operating characteristic (ROC) curve analysis and calculated the corresponding area under the curve (AUC). We compared the PDE4D7 score to individual clinical parameters and clinical models, as well as combination models incorporating the PDE4D7 score with the EAU-BCR risk score and additional clinical variables (ISUP pGleason grade group, pT Stage, extra-prostatic extension and surgical margin status, SVI, and LNI). The individual clinical parameters and clinical models demonstrated AUCs below 0.7, while the PDE4D7_clinical combination models exhibited AUCs of 0.81 (PDE4D7 EAU-BCR) and 0.87 (full risk model) (p<0.0001). These results suggest that the PDE4D7_clinical combination models have superior predictive ability for 5-year PCSM compared to the individual clinical parameters and models.
Finally, we applied Kaplan-Meier (KM) survival analysis to investigate whether pPDE4D7 percentile score classes could stratify patients receiving anti-androgens in addition to SRT into distinct subgroups with varying risks of PCSM or ACM. Our results revealed a statistically significant difference in survival outcomes between pPDE4D7 score classes (logrank p=0.0004; Figure 13D). Specifically, patients in the lower pPDE4D7 score class had a median survival of 89.6 months and an overall cancer-specific survival rate of 30% post-initiation of androgen deprivation therapy (ADT), while those in the higher pPDE4D7 score classes had not reached median survival. A similar pattern was observed for ACM, with the overall survival rate dropping below 20% for the lowest pPDE4D7 class (Figure 13E). In contrast, none of the other studied risk classification models (mxpGPS, EAU-BCR risk, CAPRA-S) exhibited the ability to stratify patients into different risk classes with significant performance in this setting.
Our data demonstrate that the PDE4D7 score is a significant predictor of disease specific (PCSM) and overall (ACM) mortality in patients with recurrent and progressive prostate cancer following primary surgical resection. Patients with higher PDE4D7 scores had a more favourable long-term survival rate, while those with lower scores exhibited a poorer prognosis in terms of survival following post-surgical treatments. These findings suggest that the PDE4D7 score may be a useful predictor of patient outcomes in this patient population.
PDE4D7 knockdown is associated with loss of androgen signaling, enrichment of the mesenchymal epithelial transition, and neuroendocrine prostate cancer.
To further investigate the biological significance of PDE4D7 in prostate cancer development and progression, we generated a derivative of the LNCaP (clone FCG) prostate cancer cell line with selective knockdown of PDE4D7 expression using a lentivirus-mediated shRNA strategy. Among several PDE4D- knockdown LNCaP clones, LNCaP Pl cells exhibited the most significant downregulation of PDE4D7 mRNA expression, as confirmed by RT-qPCR. Western blot analysis and confocal microscopy further confirmed the reduction in PDE4D7 protein expression in LNCaP Pl cells compared to the wildtype LNCaP and a scrambled shRNA control (LCNaP_SC2) (Figures 1A, IB, and 1C). These findings demonstrate the successful generation of a PDE4D7 knockdown model in LNCaP cells. We performed next-generation sequencing (NGS) RNA sequencing of the two control cell lines (LNCaP WT and LNCaP_SC2) as well as three PDE4D7 knock-down clones LNCaP Pl, LNCaP_w5.2, and LNCaP_w6.3 and used the corresponding gene expression count tables as input for gene set enrichment analysis (GSEA) of the 50 GSEA hallmark pathways (gsea-msigdb.org). For pathway analysis in phenotypes wildtype (WT) and shRNA scrambled control SC2 vs. the PDE4D7 knockdown cell lines, we identified the androgen response (AR) pathway as the most significantly enriched gene set (false discovery rate (FDR) q-value <0.001; nominal enrichment score -2.1; Figures ID, IE). To confirm that RNAseq data was reflected at the protein level, western blotting for select PCa-related proteins was performed, with significant downregulation observed in the Pl cell line compared to the WT (Figure IF). Importantly, the scrambled shRNA cell line SC2 showed no notable difference in expression of these genes. Amongst the most down-regulated genes in the two PDE4D7 knock-down cell lines were the known AR-regulated kallikrein-related peptidases KLK2 and KLK3, the transmembrane serine protease 2 (TMPRSS2), and the transcription factor NK3 homeobox-1 (NKX3-1). Also, the AR itself - although not part of this hallmark AR response pathway - is strongly downregulated in the PDE4D7 knock-down cell line Pl (Figure IF). The described findings indicate that the selective depletion of the PDE4D7 transcript expression leads to a cellular phenotype which represents androgen-insensitive proliferation.
In contrast, we identified multiple hallmark pathways significantly enriched in the PDE4D7 knock-down LNCaP derivatives. Of those, the EMT pathway demonstrated the highest level of enrichment (FDR q- value <0.001; nominal enrichment score 1.9; Figures 14, 1H). Other hallmark pathways with FDR q- values <0.1 that may further contribute to the progressive phenotype of Pl and w5.2, and are potentially relevant in a clinical setting, are those representing WNT beta catenin and hedgehog signaling. Both pathways were previously linked to progression of prostate cancer after the disease developed into hormone resistance.
NED in prostate cancer is a hallmark of aggressive, AR-independent, and treatment-resistant disease. It can occur in both primary and metastatic prostate cancer. NED is characterized by decreased expression of the AR and increased expression of neuroendocrine markers.
Given the importance of NED in aggressive prostate cancer, we evaluated a range of genes altered in NEPC for expression in our LNCaP models. This analysis revealed that many genes with induced expression in NEPC were up-regulated in the PDE4D7 knock-down models including classical markers of NED like ENO2, SYP, AURKA, or NCAMl (Figure II).
To explore the potential clinical relevance of these findings, we also analyzed expression of hallmark AR response genes and key markers for NED in RNAseq data of 533 human patient samples described previously by us and found that all tested AR response genes were down-regulated along with reduced PDE4D7 expression in these patient tumours while the AR itself did not change its expression. Similarly, we found several markers reported in NED have changed expression in low vs high PDE4D7 patient tumors and the change regulation was according to what was reported previously in NEPC.
PDE4D7-knockdown in LNCaP cells alters phenotype and confers resistance to AR antagonist Knockdown of PDE4D7 in LNCaP cell line Pl resulted in increased growth characteristics of the cells, as confirmed by real-time growth assays comparing Pl PDE4D7-knockdown cells to WT and SC2 LNCaP cells (Figure 2A). Importantly, no significant difference in growth characteristics were found between the WT and SC2 LNCaP cells.
Enzalutamide, an AR antagonist used to treat advanced PCa, was utilized to investigate the effects on growth of PDE4D7-silenced PCa cells. Dose-response experiments using real-time proliferation technology showed a significant decrease in WT LNCaP cell proliferation at all concentrations tested (0.1-30 pM), with the most profound effects at IpM and above (Figure 2B). In contrast, Pl LNCaP cells showed minimal reductions in proliferation upon treatment with enzalutamide and even exhibited increased growth at higher concentrations (Figure 2C). Re-expression of PDE4D7 in LNCaP Pl cells via a Tet-On inducible vector rescued the growth phenotype and restored sensitivity to enzalutamide (Figure 15A, 15B). This data strongly highlights that low PDE4D7 expression in PCa cells confers resistance to anti-androgens such as enzalutamide.
MR-L2, a PDE4 longform activator that binds allosterically to the PDE4D enzyme in order to mimic PKA phosphorylation in the UCR1 domain, was tested to rescue the enhanced growth phenotype caused by PDE4D7 knockdown in LNCaPs. Results showed inhibition of growth in Pl cells, but no effect on proliferation in WT LNCaPs (Figure 2E, 2F). This data suggests that activation of PDE4 in PDE4D7- knocked down LNCaPs has an inhibitory effect on their proliferative potential and highlights again the importance of PDE4D7 in driving PCa.
PDE4D7 specific knockdown in LNCaPs is associated with expression changes in DNA damage repair genes
Multiple studies have conclusively demonstrated that genomic alterations in DDR genes are prevalent in both primary and metastatic prostate cancer tissue, leading to the clinical utilization of PARP inhibitors (PARPi) in patients with DDR deficiencies. However, the emergence of resistance mechanisms has been observed in patients receiving PARPi treatment. To investigate the potential impact of PDE4D7 knockdown on DNA repair mechanisms, we utilized the REACTOME homology -directed repair of DNA double strand breaks gene set (reactome.org: R-HSA-5693538) and expanded it with additional genes from other DNA repair pathways that have been previously reported to be altered in prostate cancer. Our analysis of the expression of these genes across the LNCaP wildtype and SC2 cell lines, as well as their PDE4D7 knockdown derivatives, revealed alterations in the expression of a subset of HDR genes in the knockdown cells, particularly among DDR genes that are commonly mutated in prostate cancer. Most of these genes are significantly enhanced in expression in the PDE4D7 knock-down cells while we did not identify significant differences in single nucleotide variants between the parent vs. the knock-down cell line.
To further evaluate the correlation between low PDE4D7 expression and potential resistance to PARPi treatment, we re-analyzed previously obtained RNAseq data from 533 human clinical samples, focusing on the expression of prostate cancer-relevant DDR genes. Our analysis revealed a most significant increase in BRCA2 expression with decreasing PDE4D7 expression in patient tumours (p<0.001 (BRAC2) along with a range of other DDR related genes (Figure 16). BRCA1, ATM and BRIP1 were also upregulated but at non-significant levels (not shown). Based on these findings, we posit that the alteration of expression of DDR genes with decreasing transcription of PDE4D7 may contribute to resistance to DDR targeting with PARPi, and that cell lines with low expression of PDE4D7 following selective knockdown may exhibit resistance to PARPi compounds such as olaparib.
PDE4D7 knockdown in LNCaPs confers resistance to PARP-inhibition and is rescued upon reintroduction of PDE4D7
Olaparib is an FDA-approved PARPi that is effective against LNCaP cells. Ceralasertib is an ataxiatelangiectasia mutated and Rad3-related (ATR) inhibitor (AZD6738), which has been suggested to enhance synergistic effects in combination with olaparib in metastatic CRPC. In light of this, we wanted to determine whether PDE4D7 knockdown affects LNCaP cells response to either of these treatments. Using real-time growth analysis, LNCaP WT proliferation decreased in a dose-dependent manner with olaparib (Figure 3A), whereas the effect on PDE4D7-deficient LNCaPs was minimal (Figure 3B). Evaluation of growth rate via slope analysis yielded similar data with significant reductions in WT growth at 3, 10 and 30pM olaparib, but with unexpected reduction in slope at low concentrations (0.3 and 1 pM) in cells where PDE4D7 was knocked down. It is notable, however, that cells with reduced PDE4D7 expression were resistant to olaparib at 10 and 30pM (Figure 3B). Next, we sought to determine whether we could rescue the phenotype through transient re-introduction of PDE4D7 into the Pl cell line. As with the TET-On induction of PDE4D7 in the Pl cell line, transiently transfected Pl-PDE4D7hl®h cells revealed significant downregulation in growth (Figure 3D). Furthermore, P1-PDE4D7111811 cells were resensitised to lOpM olaparib treatment (Figure 3E), as was previously apparent in the WT LNCaP cells, with a statistically significant downregulation in growth recorded when compared to both olaparib-treated Pl cells, and DMSO-treated P1-PDE4D7+/+ cells. This data re-confirms the role of PDE4D7 is blunting of PCa cell growth and promoting susceptibility to clinically relevant PCa therapeutics.
PDE4D7-knockdown in LNCaP cells does not affect response to ATR inhibition yet may limit docetaxel treatment response
Docetaxel is a DNA-damage inducing cytotoxic drug which is clinically used in advanced prostate cancer treatment. Compared to DMSO, docetaxel significantly reduced growth of both WT and PDE4D7- knockdown LNCaPs at all concentrations, except for lOpM in Pl cells which appeared to have no effect. Given that lOpM was effective in WT but not Pl, we investigated how PDE4D7-reintroduction to Pl cells affected this. As with olaparib, P1-PDE4D7+/+ cells were more sensitive to docetaxel treatment than Pl, suggesting that a paucity of PDE4D7 promotes the ability to repair associated DNA damage and protects against induction of cell death. This is in line with the increased expression of several key DNA repair genes in PDE4D7-knockdown LNCaPs.
Interestingly, whilst PDE4D7-knockdown conferred resistance of LNCaP cells to olaparib at 10-30pM, these cells were extremely sensitive to ATR inhibition via ceralasertib at the same concentrations. Growth curves for WT and PDE4D7-knockdown LNCaP treated with increasing concentrations of ceralasertib showed similar trends with respect to sensitivity to the compound (Figures 17A, 17B, respectively). In both cases, the most significant growth inhibition occurred at 10-30pM treatment. These results highlight that PDE4D7 silencing confers resistance to PARP -inhibition via olaparib, however these cells are highly susceptible to ATR-inhibition via ceralasertib.
Given that both Ceralasertib and metformin appear to inhibit LNCaP cell growth irrespective of PDE4D7 expression status (wt vs Pl, see Examples 1 and 3) Ceralasertib or metformin may provide an alternative treatment option for prostate cancer patients with resistance to antiandrogens (such as Enzalutatmide) or PARP inhibitors (such as Olaparib), where the tumors have low expression of PDE4D7.
Table 3: Patient Demographics
Aggregated summary of the characteristics of the studied patient cohort. Demographics of the radical prostatectomy (RP) patient cohort including the N=367 patients eligible for statistical data analysis. For patient age and preoperative PSA the min and max values in the cohort are shown; median and IQR are shown in parentheses. Post-surgical pathology is given (Note: extracapsular extension was derived from pathology stage information). The outcome category illustrates the cumulative events in terms of recurrence to clinical metastases (CR), salvage radiation therapy (SRT) or androgen deprivation therapy (ADT) after surgery. Mortality is shown as prostate cancer specific mortality (PCSM) as well as overall all-cause mortality (ACM); (N/A=not available).
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Table 4: Multivariable Cox regression analysis of the PDE4D7 Score with the clinical prognostic parameters in a N=342 clinical high risk prostate cancer patient cohort. The tested endpoint was time to prostate cancer specific mortality (N=42 events). The PDE4D7 Score and the ISUP Gleason grade group were used as continuous variables; the pathology stage pT was used as categorical variable with pT2 as reference. Extra-Prostatic Extension (EPE), Seminal Vesicle Invasion (SVI), Surgical Margin Status (SMS), and Lymph Node Invasion (LNI) were used as binary variables (0=’no’; l=’yes’). The Hazard Ratio (HR), 95% confidence interval (CI) of the HR, and the logrank p-value for each survival analysis are indicated.
Figure imgf000048_0002

Claims

CLAIMS:
1. A product for use in the treatment, prevention or amelioration of prostate cancer by reducing therapy resistance in a subject in need thereof, wherein the product induces the expression of PDE4D7 or promotes activity of phosphodiesterase 4D7 and is selected from: a polynucleotide encoding a peptide having a sequence according to SEQ ID NO: 2 or a variant thereof, wherein the variant is a polynucleotide encoding a peptide having a sequence with at least 90% sequence homology with SEQ ID NO: 2 and wherein the peptide has phosphodiesterase activity; or a polynucleotide comprising a nucleotide sequence as defined in SEQ ID NO: 1 or a variant thereof, wherein the variant is a polynucleotide comprising a nucleotide sequence with at least 90% sequence homology to SEQ ID NO: 1 and wherein the polynucleotide encodes a peptide that has phosphodiesterase activity; or a peptide comprising a peptide sequence as defined in SEQ ID NO: 2 or a variant thereof, wherein the variant is a peptide comprising a peptide sequence with at least 90% sequence homology with SEQ ID NO: 2 and wherein the peptide has phosphodiesterase activity; or a peptide comprising a peptide sequence encoded by a polynucleotide comprising a nucleotide sequence as defined in SEQ ID NO: 1, or a variant thereof, wherein the variant is peptide comprising a peptide sequence encoded by a polynucleotide comprising a nucleotide sequence with at least 90% sequence homology to SEQ ID NO: 1 and wherein the polynucleotide encodes a peptide that has phosphodiesterase activity; or a compound as defined in formula I
Figure imgf000049_0001
Formula I wherein
Ri is H, (Cl-4)alkyl or (Cl-4)alkyloxy;
R2 and Re are independently selected from H and; Rs, R4 and R5 are independently selected from H, halogen, CN, (Ci-4)alkyl and (Ci-4)alkyloxy, the (Ci- 4)alkyl and (Ci-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros;
R7, Rs, and Rio are independently selected from H and F;
R9 is selected from (Cl-4)alkyl, (Cl-4)alkyloxy, CN and halogen, the (Cl-4)alkyl and (Cl-4)alkyloxy groups being optionally substituted with 1 to 3 fluoros; or a peptide having a sequence comprising or consisting a sequence selected from SEQ ID Nos: 24, 25, 26 or 27, or a polynucleotide encoding a peptide having a sequence comprising or consisting a sequence selected from SEQ ID Nos: 24, 25, 26 or Tl.
2. Product for use according to claim 1 wherein the therapy resistance is antiandrogen resistance, PARP inhibitor resistance and/or resistance to a chemotherapeutic agent, preferably wherein the antiandrogen resistance is resistance to Enzalutamide, Flutamide, Nilutamide, Bicalutamide, Topilutamide, Apalutamide, Darolutamide, Cimetidine, ketoconazole, Proxalutamide (GT-0918), Seviteronel (VT-464), or Abiraterone, more preferably resistance to Enzalutamide; and/or preferably wherein the PARP inhibitor resistance is resistance to Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, Pamiparib (BGB-290), Rucaparib, CEP 9722, or E7016, more preferably wherein the therapy resistance is resistance to Olaparib; and/or preferably wherein the resistance to a chemotherapeutic agent is resistance to a chemotherapeutic agent selected from: Paclitaxel, Docetaxel, Abraxane, Taxotere, Cabazitaxel, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Estramustine phosphate, Alestramustine, Atrimustine, Cytestrol acetate, Estradiol mustard, Estramustine, Estromustine, ICI- 85966, or Phenestrol, more preferably wherein the therapy resistance is resistance to Ocetaxel, Cabazitaxel, Mitoxantrone, Estramustine.
3. Product for use according to claim 1 or 2 wherein the therapy resistant prostate cancer has a decreased expression of PDE4D7.
4. Product for use according to any one of the preceding claims wherein the product is delivered using a nanoparticle.
5. Product for use according to any one of the preceding claims wherein the therapy resistance is antiandrogen resistance and the treatment, prevention or amelioration further comprises administering an antiandrogen, preferably an antiandrogen selected from Enzalutamide, Flutamide, Nilutamide, Bicalutamide, Topilutamide, Apalutamide, Darolutamide, Cimetidine, ketoconazole, Proxalutamide (GT-0918), Seviteronel (VT-464), or Abiraterone.
6. Product for use according to claim any one of the preceding claims wherein the therapy resistance is PARP inhibitor resistance and the treatment, prevention or amelioration further comprises administering a PARP inhibitor to the subject, preferably a PARP inhibitor selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, Pamiparib (BGB-290), Rucaparib, CEP 9722, or E7016.
7. Product for use according to claim any one of the preceding claims wherein therapy resistance is resistance to a chemotherapeutic agent and the treatment, prevention or amelioration further comprises administering a chemotherapeutic agent to the subject, preferably a chemotherapeutic agent selected from: Paclitaxel, Docetaxel, Abraxane, Taxotere, Cabazitaxel, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Estramustine phosphate, Alestramustine, Atrimustine, Cytestrol acetate, Estradiol mustard, Estramustine, Estromustine, ICI-85966, or Phenestrol.
8. Product for use according to any one of the preceding claims, wherein the product is administered to a subject in case the subject demonstrates therapy resistance or low expression of PDE4D7 in the tumor.
9. Product for use according to any one of the preceding claims, wherein the treatment, prevention or amelioration comprises providing a sample from the subject and determining the expression level of PDE4D7, preferably wherein the product is administered to the subject when a low expression level of PDE4D7 is observed in the sample
10. A compound for use in the treatment, prevention or amelioration of prostate cancer in a subject in need thereof, wherein the compound is an ATR inhibitor or an metabolic inhibitor, the use comprising administering the compound if:
- high expression of PDE4D7 is observed in a tumor;
- low expression of PDE4D7 is observed but PDE4D7 activity cannot be sufficiently increased to re-sensitize tumors to treatment with inhibitors; or
- in metastatic tumors.
11. The compound for use according to claim 10 wherein the ATR inhibitor is selected from RP-3500, Ceralasertib (AZD6738), AZ20, Elimusertib (BAY-1895344), Elimusertib (BAY-1895344) hydrochloride, SKLB-197, ETP-46464, Berzosertib (VE-822), Dactolisib (BEZ235), VE-821, Torin 2, or CGK 733, more preferably the ATR inhibitor is Ceralasertib.
12. The compound for use according to claim 10 wherein the metabolic inhibitor is selected from Methotrexate, Pemetrexed, 6-Mercaptopurine, 6-Thioguanine, Capecitabine, 5 -Fluorouracil, Gemcitabine, Cytarabine, Leflunomide, CB-839, PEG-BCT-100 (ADIPEG20), AEB-1102, L- Asparaginase, TVB-2640, AG-120 (ivosidenib), IDH305, BAY1436032, FT-2102, AG-221 (enasidenib), AG-881, AZD3965, CPI-613, or Metformin, more preferably wherein the metabolic inhibitor is metformin.
PCT/EP2023/079089 2022-10-27 2023-10-19 Treatment of prostate cancer by promoting pde4d7 WO2024088865A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP22204042.0A EP4360711A1 (en) 2022-10-27 2022-10-27 Treatment of prostate cancer by promoting pde4d7
EP22204042.0 2022-10-27
EP23155868.5 2023-02-09
EP23155868 2023-02-09

Publications (1)

Publication Number Publication Date
WO2024088865A1 true WO2024088865A1 (en) 2024-05-02

Family

ID=88511406

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2023/079089 WO2024088865A1 (en) 2022-10-27 2023-10-19 Treatment of prostate cancer by promoting pde4d7

Country Status (1)

Country Link
WO (1) WO2024088865A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120129788A1 (en) 2009-05-12 2012-05-24 The University Court Of The University Of Glasgow Phosphodiesterase 4d7 as marker for malignant, hormone-sensitive prostate cancer
EP3303618A1 (en) 2015-05-29 2018-04-11 Koninklijke Philips N.V. Methods of prostate cancer prognosis
EP3434787A1 (en) 2009-05-12 2019-01-30 Koninklijke Philips N.V. Phosphodiesterase 4d7 as prostate cancer marker

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120129788A1 (en) 2009-05-12 2012-05-24 The University Court Of The University Of Glasgow Phosphodiesterase 4d7 as marker for malignant, hormone-sensitive prostate cancer
EP3434787A1 (en) 2009-05-12 2019-01-30 Koninklijke Philips N.V. Phosphodiesterase 4d7 as prostate cancer marker
EP3303618A1 (en) 2015-05-29 2018-04-11 Koninklijke Philips N.V. Methods of prostate cancer prognosis

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
"Biocomputing: Informatics And Genome Projects", 1993, ACADEMIC PRESS
"Computer Analysis Of Sequence Data, Part I", 1994, ACADEMIC PRESS
"Genbank", Database accession no. AAN10118
"Sequence Analysis Primer", 1991, M STOCKTON PRESS
ATSCHUL, S. F. ET AL., J. MOLEC. BIOL., vol. 215, 1990, pages 403
AUSUBEL ET AL.: "Sequence Analysis In Molecular Biology", 1987, JOHN WILEY & SONS
BLAIR ET AL.: "Measuring cAMP Specific Phosphodiesterase Activity: A Two-step Radioassay", BIO PROTOC., vol. 10, no. 7, 5 April 2020 (2020-04-05), pages e3581
BOETTCHER ET AL., ONCOTARGET, vol. 7, no. 43, 25 October 2016 (2016-10-25), pages 70669 - 70684
CARILLO, H.LIPTON, D., SIAM J. APPLIED MATH, vol. 48, 1988, pages 1073
CHEN ET AL., ACTA PHARMACOL SIN, vol. 38, 2017, pages 1205 - 1235
D J P HENDERSON ET AL: "The cAMP phosphodiesterase-4D7 (PDE4D7) is downregulated in androgen-independent prostate cancer cells and mediates proliferation by compartmentalising cAMP at the plasma membrane of VCaP prostate cancer cells", BRITISH JOURNAL OF CANCER, vol. 110, no. 5, 11 February 2014 (2014-02-11), London, pages 1278 - 1287, XP055231555, ISSN: 0007-0920, DOI: 10.1038/bjc.2014.22 *
DEVEREUX, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, no. 1, 1984, pages 387
HENDERSON ET AL., BRITISH JOURNAL OF CANCER, vol. 110, 11 February 2014 (2014-02-11), pages 1278 - 1287
LEMBERG ET AL., J CLIN INVEST., vol. 132, no. 1, 4 January 2022 (2022-01-04), pages e148550
LUENGO ET AL., CELL CHEMICAL BIOLOGY, vol. 24, 21 September 2017 (2017-09-21), pages 1161 - 1180
QIU ZHAOJUN ET AL: "ATR/CHK1 inhibitors and cancer therapy", RADIOTHERAPY AND ONCOLOGY, ELSEVIER, IRELAND, vol. 126, no. 3, 18 October 2017 (2017-10-18), pages 450 - 464, XP085359683, ISSN: 0167-8140, DOI: 10.1016/J.RADONC.2017.09.043 *
RAMAMOORTH MNARVEKAR A.: "Non viral vectors in gene therapy- an overview", J CLIN DIAGN RES., vol. 9, no. 1, January 2015 (2015-01-01), pages GE01 - 6
RENÉ BÖTTCHER ET AL: "Human PDE4D isoform composition is deregulated in primary prostate cancer and indicative for disease progression and development of distant metastases", ONCOTARGET, vol. 7, no. 43, 25 October 2016 (2016-10-25), United States, pages 70669 - 70684, XP055505101, ISSN: 1949-2553, DOI: 10.18632/oncotarget.12204 *
SAMBROOK ET AL.: "Molecular Cloning. A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SMITH HANNAH L. ET AL: "DNA damage checkpoint kinases in cancer", EXPERT REVIEWS IN MOLECULAR MEDICINE, vol. 22, 8 June 2020 (2020-06-08), XP055878461, Retrieved from the Internet <URL:http://dx.doi.org/10.1017/erm.2020.3> DOI: 10.1017/erm.2020.3 *

Similar Documents

Publication Publication Date Title
Welti et al. Targeting the p300/CBP axis in lethal prostate cancer
Mancini et al. Disruption of the β1L isoform of GABP reverses glioblastoma replicative immortality in a TERT promoter mutation-dependent manner
Luo et al. Transcriptional factor specificity protein 1 (SP1) promotes the proliferation of glioma cells by up-regulating midkine (MDK)
Chu et al. Promoter methylation and downregulation of SLC22A18 are associated with the development and progression of human glioma
Ciechomska et al. Pre-treatment or post-treatment of human glioma cells with BIX01294, the inhibitor of histone methyltransferase G9a, sensitizes cells to temozolomide
Li et al. PROTAC bromodomain inhibitor ARV-825 displays anti-tumor activity in neuroblastoma by repressing expression of MYCN or c-Myc
US20210008070A1 (en) Targeting minimal residual disease in cancer with cd36 antagonists
Zhang et al. Selective inhibition of the second bromodomain of BET family proteins results in robust antitumor activity in preclinical models of acute myeloid leukemia
US20210008047A1 (en) Targeting minimal residual disease in cancer with rxr antagonists
Seo et al. Docetaxel‐resistant prostate cancer cells become sensitive to gemcitabine due to the upregulation of ABCB1
EP3752644A1 (en) Tumor minimal residual disease stratification
Zhang et al. LINC-PINT suppresses cisplatin resistance in gastric cancer by inhibiting autophagy activation via epigenetic silencing of ATG5 by EZH2
EP3092492B1 (en) Treatment of tumors expressing mutant p53
Augspach et al. Minor intron splicing is critical for survival of lethal prostate cancer
AU2016222587A1 (en) Compositions and methods of treating Fanconi Anemia
US20060252053A1 (en) Bcl2L12 polypeptide activators and inhibitors
WO2024088865A1 (en) Treatment of prostate cancer by promoting pde4d7
EP4360711A1 (en) Treatment of prostate cancer by promoting pde4d7
Mazi et al. The paracaspase MALT1 is a downstream target of Smad3 and potentiates the crosstalk between TGF-β and NF-kB signaling pathways in cancer cells
Lospinoso Severini et al. SALL4 is a CRL3REN/KCTD11 substrate that drives Sonic Hedgehog-dependent medulloblastoma
CA2833534A1 (en) Molecular subtyping, prognosis and treatment of prostate cancer
Han et al. HMGB1 knockdown increases the radiosensitivity of esophageal squamous cell carcinoma by regulating the expression of molecules involved in DNA repair
Zhong et al. BIRC6 Modulates the Protein Stability of Axin to Regulate the Growth, Stemness, and Resistance of Renal Cancer Cells via the β-Catenin Pathway
US11938164B2 (en) Exosome-based cancer assays
Zhang et al. Circ_0008285 silencing suppresses angiotensin II‐induced vascular smooth muscle cell apoptosis in thoracic aortic aneurysm via miR‐150‐5p/BASP1 axis